Cholix toxin-derived fusion moelcules for oral delivery of biologically active cargo

ABSTRACT

The present disclosure relates to pharmaceutical compositions comprising a non-naturally occurring fusion molecule and one or more pharmaceutically acceptable carriers, formulated for oral delivery to a subject, and designed to provide for improved, effective therapies for treatment of, e.g., inflammatory diseases, autoimmune diseases, cancer, metabolic disorders, and growth deficiency disorders. The present disclosure relates to a non-toxic mutant form of the  Vibrio cholera  Cholix gene (ntCholix), a variant of Cholix truncated at amino acid A 386  (Cholix 386 ) and the use of other various Cholix-derived polypeptide sequences to enhance intestinal delivery of biologically-active therapeutics. The systems and methods described herein provide for: the ability to deliver macromolecule doses without injections; the ability to deliver cargo such as siRNA or antisense molecules into intracellular compartments where their activity is required; and the delivery of nanoparticles and dendrimer-based carriers across biological membranes.

RELATED PATENT APPLICATIONS

This application claims benefit to U.S. patent application Ser. No.15/309,177, filed Nov. 4, 2016, which is a U.S. National StageApplication pursuant to 35 U.S.C. §371 of PCT/US2015/029795, filed May7, 2015, which claims benefit of U.S. Provisional Application No.61/990,054, filed on May 7, 2014, each incorporated in its entirety byreference herein.

TECHNICAL FIELD

The field of the present invention relates, in part, to a strategy fornovel pharmaceutical applications. More specifically, the presentinvention relates to a non-toxic mutant form of the Vibrio choleraCholix gene (ntCholix), a variant of Cholix truncated at amino acid A³⁸⁶(Cholix³⁸⁶) and the use of other various Cholix-derived polypeptidesequences to enhance intestinal delivery of biologically-activetherapeutics. Importantly, the systems and methods described hereinprovide for the following: the ability to deliver macromolecule doseswithout injections; the ability to deliver cargo, such as (but notlimited to) siRNA or antisense molecules into intracellular compartmentswhere their activity is required; and the delivery of nanoparticles anddendrimer-based carriers across biological membranes, which otherwisewould have been impeded due to the barrier properties of most suchmembranes.

Oral delivery of biologically active polypeptides (referring to apolymer composed of amino acid residues; typically also defined asproteins or peptides) has been a long-standing goal of thepharmaceutical industry. Unfortunately, the numerous physical,physiological, and biological barriers of the gastrointestinal (GI)tract are designed to inhibit uptake of proteins and peptides until theycan be sufficiently degraded for absorption through amino acid and di-or tri-peptide transporters; and/or to traffic the proteins and peptidesintracellularly to destructive lysosome compartments after endosomaluptake at the luminal surface. As such, the feasibility of polypeptideuptake from the intestine in a manner similar to that achievable with,e.g., small molecules, has been limited and low oral bioavailabilitycontinues to be a problem for most polypeptides and proteins.

While there have been some promising results from clinical studiesevaluating various biologically active polypeptides for the treatment ofdiseases such as cancer, inflammatory diseases, immune diseases, growthdeficiency disorders, etc., and several DNA-based therapeutics have beenFDA approved for such uses, these therapeutics often fail to reallyreach their optimum potential, as there is often marginal or inadequateoverall efficacy due to inherent limitations such as short biologicalhalf-life which prevents the delivery of optimal therapeuticallyeffective dosages, and/or detrimental side effects and toxicitiesobserved at the therapeutically effective doses. Moreover, many suchtherapeutics require multiple dosing regimens, necessitating continuousadministration intravenously or by frequent subcutaneous injections,which are burdensome on the patients and caregivers.

Future clinical studies directed toward evaluating the promisingbiologically active polypeptides could benefit greatly from new methodsand/or pharmaceutical compositions that could be used to orallyadminister such polypeptides to a human subject.

BACKGROUND ART

The majority of currently-approved small molecule drugs are absorbedacross the mucosa of the small intestine to provide delivery to thesystemic circulation. In fact, small molecule drugs are selected basedupon their stability and efficient absorption across intestinal mucosae.A similar oral delivery of biologically-active polypeptides (referringto a polymer composed of amino acid residues; typically defined as aprotein or peptide) has been a long-standing goal of the pharmaceuticalindustry. As the gastrointestinal (GI) tract is designed to digestdietary proteins and peptides, there are numerous physical,physiological, and biological barriers that limit the feasibility oftherapeutic proteins and peptides uptake from the intestine in a mannersimilar to that achievable with small molecules; Mahato, R. I., et al.,Crit Rev Ther Drug Carrier Syst, 20(2-3):p. 153-214 (2003).

A number of technologies have been identified that can be used toprotect therapeutic proteins and peptides through the stomach, allowingthem to reach the absorptive surface of epithelial cells in the smallintestine and separating them from the gastric and intestinalenvironments that function to destroy dietary proteins and peptides.Unfortunately, however, the efficient transport across this simple,single layer of cells remains a substantial barrier due to theintracellular trafficking to destructive lysosome compartments afterendosomal uptake of polypeptides at the luminal surface; Woodley, J. F.,Crit Rev Ther Drug Carrier Syst, 11(2-3):p. 61-95 (1994). Indeed, thisbarrier is designed to inhibit uptake of proteins and peptides untilthese macromolecules can be sufficiently degraded for absorption throughamino acid and di- or tri-peptide transporters. In this regard, a numberof efforts have been examined to overcome the physical, physiological,and biological barriers of the intestinal mucosae.

There are two basic routes across the simple epithelium that constitutesthe cellular barrier of the intestinal mucosae. Specifically, onceacross the covering mucus layer, a molecule could move between adjacentepithelial cells (paracellular route) or move through cells(transcellular route) via a series of vesicles that traffic within, butdo not mingle, contents with the cytoplasm; T. Jung et al., Eur J PharmBiopharm, 50:147-160 (2000). In other words, in both routes, a transportprotein or peptide therapeutic does not enter into the cell but ratherstays in an environment external to the cell's cytoplasm.

The primary barrier to casual movement of therapeutic protein andpeptide movement through the paracellular route is a complex of proteinsat the apical neck of these cells known as the tight junction (TJ).While transient opening and closing of TJ structures can facilitatetransport of peptides across intestinal epithelia, this approach has keylimitations: e.g., it does not work well for molecules above ˜5 kDa; ithas the potential for non-selective entry of materials into the bodyfrom the intestinal lumen; and it represents a route that involves onlya small fraction of the surface area of the intestinal epithelium.

The primary barrier to casual migration of protein or peptidetherapeutics across cells via the transcellular route is a defaultvesicle trafficking that delivers the contents of these vesicles to adestructive (lysosomal) pathway. As compared to the paracellular route,movement through the vesicular transcellular route can accommodatematerials as large as 100 nm in diameter, involves essentially theentire epithelial cell surface, and can be highly selective in uptake ofmaterials through the use of receptor-ligand interactions for vesicleentry. Thus, the transcellular route is very appealing for theepithelial transport of protein or peptide therapeutics if thedestructive pathway can be avoided.

Some pathogens have solved the trafficking barrier problem, asdemonstrated by the efficient transcytosis of secreted polypeptidevirulence factors which function to facilitate and/or stabilizeinfection of a host. Exotoxins represent a class of proteins released bya variety of microorganisms which function as potent virulence factors.Exotoxins function on multi-cellular organisms with the capacity to actsas potent toxins in man; Roszak, D. B., and Colwell, R. R., MicrobiolRev 51:365-379 (1987). These proteins commonly kill or inactivate hostcells through mechanisms that involve selective disruption of proteinsynthesis. Accordingly, only a few molecules are required to kill,consistent with the observation that bacterial exotoxins are some of themost toxic agents known. A subset of these proteins comprised of thefamily of proteins that consists of diphtheria toxin (DT) fromCorynebacterium diphtheria, exotoxin A from Pseudomonas aeruginosa (PE),and a recently identified protein termed Cholix from Vibrio cholerafunction to intoxicate host cells via the ADP-ribosylation of elongationfactor 2 (EF2); Yates, S. P., et al., Trends Biochem Sci, 31:123-133(2006). These exotoxins are synthesized as a single chain of amino acidsthat fold into distinct domains that have been identified as havingspecific functions in targeting, entry, and intoxication of host cells.

The biology of exotoxin A from Pseudomonas aeruginosa (PE) has recentlybeen described; Mrsny, R. J., et al., Drug Discov Today, 7(4): p. 247-58(2002). PE is composed of a single chain of 613 amino acids having atheoretical molecular weight (MW) of 66828.11 Da, an isoelectric point(pI) of 5.28, and that functionally folds into three discrete domains,denoted domain I (Ala¹-Glu²⁵²), domain II (Gly²⁵³-Asn³⁶⁴), domain III(Gly⁴⁰⁵-Lys⁶¹³, and which contains a ADP-ribosyltransferase activitysite), and a short disulfide-linked loop linking domains II and IIIwhich is known as the Ib loop (Ala365-Gly404). The organization of thesedomains at pH 8.0 have determined from crystal diffraction at aresolution of ˜1.5 Å; Wedekind, J. E. et al., J Mol Biol, 314:823-837(2001). Domain I (Ia+Ib) has a core formed from a 13-stranded β-roll,domain II is composed of six α-helices, and domain III has a complexα/β-folded structure. Studies have supported the idea that the modularnature of PE allows for distinct domain functions: domain I binds tohost cell receptors, domain II is involved in membrane translocation,and domain III functions as an ADP-ribosyltransferase. It appears thatPE is secreted by P. aeruginosa in close proximity to the epithelialcell apical surface, possibly in response to environmental cues and/orcellular signals; Deng, Q. and J. T. Barbieri, Annu Rev Microbiol, 62:p.271-88 (2008). Once secreted, internalization into cells occurs afterdomain I of PE binds to the membrane protein a2-macroglobulin, a proteinwhich is also known as the low-density lipoprotein receptor-relatedprotein 1 (LRP1) or CD91; see, e.g., FitzGerald, D. J., et al., J CellBiol, 129(6):p. 1533-41 (1995); Kounnas, M. Z., et al., J Biol Chem,267(18): p. 12420-3 (1992). Following internalization, PE avoidstrafficking to the lysosome and is instead efficiently delivered to thebasolateral surface of the cell where it is released in abiologically-active form; Mrsny, R. J., et al., Drug Discov Today, 7(4):p. 247-58 (2002). Once across the epithelium, PE functions as avirulence factor by entering into CD91-positive cells within thesubmucosal space (macrophage and dendritic cells) where it thenintersects with an unfolding pathway that leads to the cytoplasmicdelivery of domain III; see, e.g., Mattoo, S., Y. M. Lee, and J. E.Dixon, Curr Opin Immunol, 19(4): p. 392-401 (2007); Spooner, R. A., etal., Virol J, 3: p. 26 (2006).

Vibrio cholerae bacterium is best known for its eponymous virulenceagent, cholera toxin (CT), which can cause acute, life-threateningmassive watery diarrhea. CT is a protein complex composed of a single Asubunit organized with a pentamer of B subunits that binds to cellsurface G_(M1) ganglioside structures at the apical surface ofepithelia. CT is secreted by V. cholera following horizontal genetransfer with virulent strains of V. cholerae carrying a variant oflysogenic bacteriophage called CTXf or CTXφ. Recent cholera outbreaks,however, have suggested that strains of some serogroups (non-O1,non-O139) do not express CT but rather use other virulence factors.Detailed analyses of non-O1, non-O139 environmental and clinical datasuggested the presence of a novel putative secreted exotoxin with somesimilarity to PE.

Jorgensen, R. et al., J Biol Chem, 283(16):10671-10678 (2008) reportedthat some strains of V. cholerae did, in fact, contain a protein toxinhaving similarity to PE and which they termed Cholix toxin (Cholix).Compared to PE, Cholix has a slightly larger theoretical MW (70703.89Da) and a slightly more acidic theoretical pI (5.12). The crystalstructure of the 634 amino acid Cholix protein has been resolved to ˜2Å. The domain structure and organization was found to be somewhatsimilar to PE: domain I (Val¹-Lys²⁶⁵), domain II (Glu²⁶⁶-Ala³⁸⁶), domainIII (Arg⁴²⁶-Lys⁶³⁴), and a Ib loop (Ala³⁸⁷-Asn⁴²⁵). Additionalstructural similarity to PE includes: a furin protease site for cellularactivation; a C-terminal KDEL sequence that can route the toxin to theendoplasmic reticulum of the host cell; and an ADP-ribosyltransferaseactivity site within domain III.

Remarkably, PE and Cholix share no significant genetic and limitedsimilarity by amino acid alignment. Searching the genome of V. cholerafor PE-like nucleotide sequences fails to result in a match of any kind.It is only at the protein sequence level is there the hint that anPE-like protein could be produced by this bacterium. Even here, there isonly a 32% homology between the amino acid sequences of PE and Cholixwith similarities (42% homology) being focused in the ADP ribosylationelements of domain III, and with low levels of amino acid homology(˜15-25%) for most segments of domains I and II for the two proteins.Moreover, this overall arrangement of Cholix relative to PE is even morestriking since these two proteins with similar elements were derivedfrom two distinct directions: P. aeruginosa is a GC-rich bacterium whileV. cholera is AT-rich. That these two toxins evolved from two differentgenetic directions to arrive at nearly the same structure but with only32% amino acid homology suggests that structural and functionalsimilarities of Cholix and PE are likely based upon similar survivalpressures rather than through similar genetic backgrounds. The very lowamino acid homology of domains I and II for these two proteins stressthe functional importance of the folded structures of these two proteinsand not their amino acid sequences.

The C-terminal portion of Cholix and PE appear to function in theintoxication of cells through ADP-ribosylation of EF2 in comparableways. Recent studies where the latter half of Cholix (domain I deleted)targeted to cancer cells through conjugation to an antibody directed tothe transferrin receptor suggests that the C-terminal portions of PE andCholix involved in ADP-ribosylation of EF2 are indeed functionallysimilar; Sarnovsky, R., et al., Cancer Immunol Immunother59:737-746(2010). While this distal portion of Cholix is 36% identical and 50%similar to PE, polyclonal antisera raised in animals as well as serafrom patients having neutralizing immune responses to this same distalportion of PE failed to cross-react with this latter portion of Cholix.Similarly, antisera raised to this Cholix failed to cross-react with PE.This data suggests that while both PE and Cholix share a capacity tointoxicate cells through a similar mechanism and that these two proteinsshare a common core structure, there are striking differences in theirelements that are expressed at the surface of these proteins.

As previous studies using PE have demonstrated that this toxin readilytransports across polarized monolayers of epithelial cells in vitro andin vivo without intoxication; Mrsny, R. J., et al., Drug Discov Today,7(4): p. 247-58 (2002), the present inventors have commenced research tofurther evaluate the properties and biology of Cholix, with a particularfocus on the functional aspects of the proximal portions of Cholix;specifically, the use of domains I and II to facilitate transport acrossintestinal epithelial monolayers. As domains I and IIa appeared to bethe only essential elements of PE required for epithelial transcytosis,it was particularly important to examine these same domains in Cholix.As stated previously, there is only ˜15% -25% amino acids homology overmost of the regions that would be considered to be part of domains I andIIa. The present inventors examined the domains though a series ofstudies: monitoring the biological distribution of Cholix followingapplication to epithelial surfaces in vivo, assessment of Cholixtranscellular transport characteristics across polarized epithelial cellmonolayers in vitro, and delivery of a biologically-active cargogenetically integrated into the Cholix protein at its C-terminus.Preliminary data generated by genetically fusing the first two domainsof Cholix (amino acids 1-386) to green fluorescent protein (GFP) orchemically coupling these expressed domains to 100 nm diameter latexbeads demonstrated that Cholix attached to 100 nm latex beads wereobserved to transport across intestinal epithelial monolayers in vitroand in vivo. That the GFP cargo retained its fluorescent characterduring and after the transcytosis process also support the contentionthat Cholix utilizes a non-destructive (or privileged) traffickingpathway through polarized epithelial cells. This outcome bodes well forits (repeated) application as a tool to deliver biologically activecargos across epithelial barriers of the body, such as those in therespiratory and gastrointestinal tracts.

Also of important note from the preliminary studies is the observationwhich suggests an apparent cell receptor interaction difference betweenPE and Cholix. As stated previously, PE enters into epithelial cellsafter domain I of PE binds to the membrane protein α2-macroglobulin, aprotein which is also known as the low-density lipoproteinreceptor-related protein 1 (LRP1) or CD91. While the exact identity ofthe surface receptor for Cholix has not been established, preliminarystudies suggest that Cholix does not intoxicate some cell lines thatexpress CD91 but intoxicates some cell lines that do express CD91. It iscurrently unclear what other receptors, beyond CD91, might be involvedepithelial transcytosis of PE. Nevertheless, Cholix and PE appear tohave distinct cell receptor interactions, demonstrating cleardifferences that are sufficient to suggest very different andunanticipated applications for both oral biologics and the intracellulardelivery of bioactive agents.

DISCLOSURE OF THE INVENTION

The present invention is based on the membrane-trafficking properties ofCholix and the demonstration that Cholix transports efficiently acrosspolarized epithelial cells of the airway and intestine, suggesting thatCholix-derived polypeptide sequences (including the proximal elements ofthe protein) can be used for the efficient transcytosis of protein andnanoparticles, representing a strategy for novel pharmaceuticalapplications.

As such, one aspect of the present invention is to provide isolateddelivery constructs (e.g., genetic fusions or chemical constructs)comprising a transporter domain (e.g., a Cholix-derived polypeptidesequence) and a cargo. Both the transport domain and cargo may beexpressed/linked in varying stoichiometric ratios and spatialorganization. Different cargos may also be expressed/linked on the sameconstruct. In preferred embodiments such cargo may include one, or anyof: proteins, peptides, small molecules, siRNA, PNA, miRNA, DNA,plasmid, and antisense.

Another aspect of the present invention is to provide for the ability todeliver cargo, such as macromolecules, without injections.

Another aspect of the present invention is to provide for the ability todeliver cargo, such as (but not limited to) macromolecules, smallmolecules, siRNA, PNA, miRNA, DNA, plasmid and antisense molecules, intointracellular compartments where their activity is required.

Another aspect of the present invention is to provide for the transportof cargo via delivery of nanoparticles and/or dendrimer-based carriersacross biological membranes.

Methods of administration/delivery contemplated for use in the presentinvention include, e.g., oral administration, pulmonary administration,intranasal administration, buccal administration, sublingualadministration, ocular administration (including, but not limited to,delivery to the vitreous, cornea, conjunctiva, sclera, and posterior andanterior compartments of the eye), topical application, injection(needle or needle-free), intravenous infusion, microneedle application,administration via a drug depot formulation, administration viaintrathecal application, administration via intraperitoneal application,administration via intra-articular application, deliveryintracellularly, delivery across blood brain barrier, delivery acrossblood retina barrier, administered for local delivery and action, and/ordelivered for systemic delivery.

In yet another aspect, the invention provides a pharmaceuticalcomposition comprising the delivery constructs and a pharmaceuticallyacceptable carrier.

The present disclosure relates to pharmaceutical compositions comprisingnovel, non-naturally occurring fusion molecules and one or morepharmaceutically acceptable carriers, formulated for oral delivery, anddesigned to provide for improved, effective therapies for treatment of,e.g., inflammatory diseases and/or autoimmune diseases and/or cancers.

The present disclosure is based in part on the inventors' unique insightthat oral delivery of a pharmaceutical composition comprising a fusionmolecule which comprises a modified Cholix toxin coupled to abiologically active cargo may, among other things, provide the followingadvantages: a) in embodiments wherein the modified Cholix toxin iscoupled to the biologically active cargo without a linker, or with anon-cleavable linker, the anchoring effect of the modified Cholix toxinby its receptor(s) at the surface of, e.g., immune cells that alsoexpress the receptor for the biologically active cargo, can allow forgreater exposure of the biologically active cargo at the surface of thetargeted cells and provide a synergistic effect by binding to both theCholix receptor and the biologically active cargo receptor; b) inembodiments wherein the modified Cholix toxin is coupled to thebiologically active cargo with a linker that is cleavable by an enzymepresent at a basolateral membrane of an epithelial cell, or an enzymepresent in the plasma of the subject, such cleavage will allow thebiologically active cargo to be released from the remainder of thefusion molecule soon after transcytosis across the epithelial membranec) the direct delivery of the biologically active cargo to thesubmucosal-GI space and hepatic-portal system may reduce the systemictoxicity observed when the cargo are administered by parenteral routes,as well as enabling access to the submucosal target biology that wasdifficult to target via non-oral or GI routes; d) once transportedacross the GI epithelium, the fusion molecules of the disclosure willexhibit extended half-life in serum, that is, the biologically activecargo of the fusion molecules will exhibit an extended serum half-lifecompared to the biologically active cargo in its non-fused state; e)oral administration of the fusion molecule can deliver a highereffective concentration of the delivered biologically active cargo tothe liver of the subject than is observed in the subject's plasma; andf) the ability to deliver the biologically active cargo to a subjectwithout using a needle to puncture the skin of the subject, thusimproving such subjects' quality of life by avoiding pain or potentialcomplications associated therewith, in addition to improvedpatient/care-giver convenience and compliance.

Thus, in one aspect, the present disclosure relates to pharmaceuticalcompositions comprising a non-naturally occurring fusion molecule andone or more pharmaceutically acceptable carriers, formulated for oraldelivery, wherein the fusion molecule comprises a modified Cholix toxincoupled to a biologically active cargo to be delivered to a subject,wherein the Cholix toxin is non-toxic.

In one aspect, the present disclosure relates to pharmaceuticalcompositions comprising a non-naturally occurring fusion molecule andone or more pharmaceutically acceptable carriers, formulated for oraldelivery, wherein the fusion molecule comprises a modified Cholix toxincoupled to a biologically active cargo to be delivered to a subject,wherein the Cholix toxin is non-toxic, and wherein the fusion moleculehas the ability to activate the receptor for the biologically activecargo, or to enable the catalytic process of a catalytically-activematerial.

In various embodiments, the fusion molecules of the pharmaceuticalcompositions comprise a modified Cholix toxin truncated at an amino acidresidue within Cholix toxin domain II. In various embodiments, thefusion molecules comprise a truncated Cholix toxin having the amino acidsequence set forth in, e.g., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40 or SEQ ID NO: 41.

In various embodiments, the fusion molecules of the pharmaceuticalcompositions comprise a modified Cholix toxin truncated at an amino acidresidue within Cholix toxin domain Ib. In various embodiments, thefusion molecules comprise a truncated Cholix toxin having the amino acidsequence set forth in, e.g., SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:48, SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80.

In various embodiments, the fusion molecules of the pharmaceuticalcompositions comprise a modified Cholix toxin wherein domain III hasbeen truncated or mutated. In various embodiments, the fusion moleculescomprise a mutated Cholix toxin having the amino acid sequence set forthin SEQ ID NO: 81 wherein the amino acid residue E581 of SEQ ID NO: 1 hasbeen deleted (designated herein as “Cholix ΔE581”).

In various embodiments, the fusion molecules of the pharmaceuticalcompositions comprise a modified Cholix toxin wherein domain Ia has beenmutated.

In various embodiments, the biologically active cargo is selected frome.g., a macromolecule, small molecule, peptide, polypeptide, nucleicacid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA,plasmid, vaccine, polymer nanoparticle, or catalytically-activematerial.

In various embodiments, the biologically active cargo is an enzymeselected from hyaluronidase, streptokinase, tissue plasminogenactivator, urokinase, or PGE-adenosine deaminase.

In various embodiments, the biologically active cargo is a polypeptidethat is a modulator of inflammation in the GI tract selected from, e.g.,interleukin-10, interleukin-19, interleukin-20, interleukin-22,interleukin-24, or interleukin-26. In various embodiments, thebiologically active polypeptide is interleukin-10 having the amino acidsequence set forth is SEQ ID NO: 82. In various embodiments, thebiologically active polypeptide is interleukin-19 having the amino acidsequence set forth is SEQ ID NO: 83. In various embodiments, thebiologically active polypeptide is interleukin-20 having the amino acidsequence set forth is SEQ ID NO: 84. In various embodiments, thebiologically active polypeptide is interleukin-22 having the amino acidsequence set forth is SEQ ID NO: 85. In various embodiments, thebiologically active polypeptide is interleukin-24 having the amino acidsequence set forth is SEQ ID NO: 86. In various embodiments, thebiologically active polypeptide is interleukin-26 having the amino acidsequence set forth is SEQ ID NO: 87. In various embodiments, thebiologically active cargo is a modulator of inflammation in the GI tractthat is a small molecule. In various embodiments, the biologicallyactive cargo is a modulator of inflammation in the GI tract that is anantisense or siRNA molecule.

In various embodiments, the biologically active cargo is a TNFSFinhibitor that is an antibody, or a fragment thereof, or an artificialconstruct comprising an antibody or fragment thereof, or an artificialconstruct designed to mimic the binding of an antibody or fragmentthereof to its antigen. In various embodiments, the biologically activecargo is a TNFSF inhibitor that is a soluble TNFSF receptor fusionprotein. In various embodiments, the biologically active cargo is aTNFSF inhibitor that is a small molecule. In various embodiments, thebiologically active cargo is a TNFSF inhibitor that is an antisense orsiRNA molecule.

In various embodiments, the biologically active cargo is an antibodycomprising the heavy chain variable region amino acid sequence set forthin SEQ ID NO: 88 and light chain variable region amino acid sequence setforth in SEQ ID NO: 89. In various embodiments, the biologically activecargo is an antibody comprising the heavy chain variable region aminoacid sequence set forth in SEQ ID NO: 90 and light chain variable regionamino acid sequences set forth in SEQ ID NO: 91. In various embodiments,the biologically active cargo is a soluble TNFSF receptor fusion proteindimer comprising the amino acid sequence set forth in SEQ ID NO: 92.

In one aspect, the present disclosure relates to pharmaceuticalcompositions comprising novel, non-naturally occurring fusion moleculesand one or more pharmaceutically acceptable carriers, formulated fororal delivery, and designed to provide for improved, effective therapiesfor treatment of metabolic disorders, e.g., Type 1 Diabetes and Type 2Diabetes. Oral delivery of biologically active polypeptides (referringto a polymer composed of amino acid residues; typically also defined asproteins or peptides) has been a long-standing goal of thepharmaceutical industry. Unfortunately, the numerous physical,physiological, and biological barriers of the gastrointestinal (GI)tract are designed to inhibit uptake of proteins and peptides until theycan be sufficiently degraded for absorption through amino acid and di-or tri-peptide transporters; and/or to traffic the proteins and peptidesintracellularly to destructive lysosome compartments after endosomaluptake at the luminal surface. As such, the feasibility of polypeptideuptake from the intestine in a manner similar to that achievable with,e.g., small molecules, has been limited and low oral bioavailabilitycontinues to be a problem for most polypeptides and proteins.

In various embodiments, the present disclosure relates to pharmaceuticalcompositions comprising a non-naturally occurring fusion molecule andone or more pharmaceutically acceptable carriers, formulated for oraldelivery, wherein the fusion molecule comprises a modified Cholix toxincoupled to a glucose-lowering agent to be delivered to a subject.

In various embodiments, the present disclosure is based in part on thatoral delivery of a pharmaceutical composition comprising a fusionmolecule which comprises a modified Cholix toxin coupled to aglucose-lowering agent may, among other things, provide the followingadvantages: a) in embodiments wherein the modified Cholix toxin iscoupled to the glucose-lowering agent without a linker, the anchoringeffect of the modified Cholix toxin by its receptor(s) at the surface ofcells that also express the receptor for the glucose-lowering agent, canallow for greater exposure of the glucose-lowering agent at the surfaceof the targeted cells; b) in embodiments wherein the modified Cholixtoxin is coupled to the glucose-lowering agent with a linker that iscleavable by an enzyme present at a basal-lateral membrane of anepithelial cell, or an enzyme present in the plasma of the subject, suchcleavage will allow the glucose-lowering agent to be released from theremainder of the fusion molecule soon after transcytosis across theepithelial membrane; c) the direct delivery of the glucose-loweringagent to the submucosal-GI space and hepatic-portal system may reducethe systemic toxicity observed when the glucose-lowering agents areadministered by parenteral routes, as well as enabling access to thesubmucosal target biology that was difficult to target via non-oral orGI routes; d) the direct delivery of the glucose-lowering agent to thesubmucosal-GI space and hepatic-portal system may provide for improveddosing regimens, including less frequent insulin injections; and e) theability to deliver the glucose-lowering agent to a subject without usinga needle to puncture the skin of the subject, thus improving suchsubjects' quality of life by avoiding pain or potential complicationsassociated therewith.

In various embodiments, the glucose-lowering agent is selected frome.g., a macromolecule, small molecule, peptide, polypeptide, nucleicacid, mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA,plasmid, vaccine, polymer nanoparticle, or catalytically-activematerial. In various embodiments, the glucose-lowering agent is anincretin or incretin mimetic. In various embodiments, theglucose-lowering agent is a GLP-1. In various embodiments, theglucose-lowering agent is a GLP-1 agonist. In various embodiments, theglucose-lowering agent is an exendin. In various embodiments, theglucose-lowering agent is a glucose inhibitory protein receptor (GIPR)agonist.

In various embodiments, the glucose-lowering agent is a GLP-1 agonistthat is a peptide. In various embodiments, the glucose-lowering agent isa GLP-1 agonist that is a small molecule. In various embodiments, theglucose-lowering agent is a GLP-1 agonist that is an antisense or siRNAmolecule. In various embodiments, the glucose-lowering agent is a GLP-1agonist that is an antibody, or a fragment thereof, or an artificialconstruct comprising an antibody or fragment thereof, or an artificialconstruct designed to mimic the binding of an antibody or fragmentthereof to its antigen.

In various embodiments, the biologically active cargo is aglucose-lowering agent that is a GLP-1 agonist peptide comprising theamino acid sequence set forth in SEQ ID NO: 93. In various embodiments,the biologically active cargo is a glucose-lowering agent that is aGLP-1 agonist peptide comprising the amino acid sequence set forth inSEQ ID NO: 94.

In one aspect, the present disclosure relates to pharmaceuticalcompositions comprising novel, non-naturally occurring fusion moleculesand one or more pharmaceutically acceptable carriers, formulated fororal delivery, and designed to provide for improved, effective therapiesfor treatment of growth hormone deficiency, and like disorders.

In various embodiments, the present disclosure relates to pharmaceuticalcompositions comprising a non-naturally occurring fusion molecule andone or more pharmaceutically acceptable carriers, formulated for oraldelivery, wherein the fusion molecule comprises a modified Cholix toxincoupled to a growth hormone (GH) to be delivered to a subject.

In various embodiments, the present disclosure is based in part on theinventors' unique insight that oral delivery of a pharmaceuticalcomposition comprising a fusion molecule which comprises a modifiedCholix toxin coupled to a growth hormone may, among other things,provide the following advantages: a) in embodiments wherein the modifiedCholix toxin is coupled to the growth hormone with a linker that iscleavable by an enzyme present at a basolateral membrane surface of anepithelial cell, or an enzyme present in the plasma of the subject, suchcleavage will allow the growth hormone to be released from the remainderof the fusion molecule soon after transcytosis across the epithelialmembrane; b) the direct delivery of the growth hormone to thesubmucosal-GI space and hepatic-portal system may reduce systemictoxicities observed when the growth hormones are administered byparenteral routes, as well as enabling access to the submucosal targetbiology that was difficult to target via non-oral or GI routes (e,g,provide a more efficient induction of IGF-1 relative to systemicdelivery via subcutaneous (sc) injection); c) the direct delivery of thegrowth hormone to the submucosal-GI space and hepatic-portal system mayprovide for improved dosing regimens; d) oral delivery will achieve abrief pulse of growth hormone to the liver that is more consistent withserum level observed in growing children, and this pulse profile is notachievable by sc injection; and e) the ability to deliver the growthhormone to a subject without using a needle to puncture the skin of thesubject, thus improving such subjects' quality of life by avoiding painor potential complications associated therewith, in addition to improvedpatient/care-giver convenience and compliance.

In various embodiments, the growth hormone is selected from e.g., amacromolecule, small molecule, peptide, polypeptide, nucleic acid, mRNA,miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid,vaccine, polymer nanoparticle, or catalytically-active material. Invarious embodiments, the growth hormone is human growth hormone (or avariant thereof), growth hormone 2, or growth hormone-releasing hormone.In various embodiments, the growth hormone is human growth hormone(somatotropin) comprising the amino acid sequence set forth in SEQ IDNO: 95.

In various embodiments, the fusion molecules comprise a modified Cholixtoxin directly coupled to a biologically active cargo. In variousembodiments, the biologically active cargo is directly coupled to theC-terminus of the Cholix toxin.

In various embodiments, the fusion molecules comprise a modified Cholixtoxin chemically coupled to a biologically active cargo.

In various embodiments, the fusion molecules comprise a Cholix toxincoupled to a biologically active cargo by a non-cleavable linker. Invarious embodiments, the non-cleavable linker comprises the amino acidsequence of, e.g., SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 or SEQ IDNO: 99.

In various embodiments, the fusion molecules comprise a Cholix toxincoupled to a biologically active cargo by a cleavable linker. In variousembodiments, the linker is cleavable by an enzyme that is present at abasolateral membrane of a polarized epithelial cell of the subject. Invarious embodiments, the linker is cleavable by an enzyme that ispresent in the plasma of said subject. In various embodiments, thecleavable linker comprises the amino acid sequence of, e.g., SEQ ID NO:100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO:118, SEQ ID NO: 119, or SEQ ID NO: 120.

In various embodiments, the fusion molecules comprise a Cholix toxincoupled to a biologically active cargo by a cleavable linker, whereinthe cleavable linker comprises an amino acid sequence that is known tobe a substrate for tobacco etch virus (TEV) protease. In variousembodiments, the cleavable linker comprises the amino acid sequence of,e.g., SEQ ID NO: 121.

In various embodiments, the fusion molecule comprises the amino acidsequence set forth in SEQ ID NO: 122. (this is Cholix⁴¹⁵-TEV-IL-10)

In various embodiments, the fusion molecule comprises the amino acidsequence set forth in SEQ ID NO: 123. (this is Cholix⁴¹⁵-(G₄5)₃-IL-10).

In another aspect, the present disclosure provides a method of treatingan inflammatory disease in a subject, comprising orally administering apharmaceutical composition of the present disclosure to the subject. Invarious embodiments, the inflammatory disease is selected from aninflammatory bowel disease, psoriasis or bacterial sepsis. In variousembodiments, the inflammatory bowel disease is Crohn's disease,ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemiccolitis, diversion colitis, Behcet's syndrome or indeterminate colitis.

In another aspect, the present disclosure provides a method of treatingan autoimmune disease in a subject, comprising orally administering apharmaceutical composition of the present disclosure to the subject. Invarious embodiments, the autoimmune disease is systemic lupuserythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolyticanemia, thrombocytopenia purpura, Grave's disease, Sjogren's disease,dermatomyositis, Hashimoto's disease, polymyositis, inflammatory boweldisease, multiple sclerosis (MS), diabetes mellitus, rheumatoidarthritis, or scleroderma.

In another aspect, the present disclosure provides a method of treatinga cancer in a subject, comprising orally administering a pharmaceuticalcomposition of the present disclosure to the subject. In variousembodiments, the cancer to be treated includes, but is not limited to,non-Hodgkin's lymphomas, Hodgkin's lymphoma, chronic lymphocyticleukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiplemyeloma, carcinomas of the bladder, kidney ovary, cervix, breast, lung,nasopharynx, malignant melanoma and rituximab resistant NHL andleukemia.

In another aspect, the present disclosure provides a method of treatinga subject having a metabolic disorder, said method comprising orallyadministering a fusion molecule of the present disclosure in an amountsufficient to treat said disorder, wherein said metabolic disorder isdiabetes, obesity, diabetes as a consequence of obesity, hyperglycemia,dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance,impaired glucose tolerance (IGT), diabetic dyslipidemia, orhyperlipidemia.

In another aspect, the present disclosure provides a method of treatinga subject having a fatty liver disease (e.g., nonalcoholic fatty liverdisease (NAFLD); nonalcoholic steatohepatitis (NASH)), agastrointestinal disease, or a neurodegenerative disease, said methodcomprising orally administering a fusion molecule of the presentdisclosure in an amount sufficient to treat said disease.

In another aspect, the present disclosure provides a method of treatinga subject having a GH deficient growth disorder, said method comprisingorally administering a fusion molecule of the present disclosure in anamount sufficient to treat said disorder, wherein said disorder isgrowth hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome,Prader-Willi syndrome, short stature homeobox-containing gene (SHOX)deficiency, chronic renal insufficiency, and idiopathic short statureshort bowel syndrome, GH deficiency due to rare pituitary tumors ortheir treatment, and muscle-wasting disease associated with HIV/AIDS.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof an inflammatory disease in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof an autoimmune disease in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof a cancer in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof a metabolic disorder in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof a fatty liver disease in a subject in need thereof.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present invention for thepreparation of a medicament for treatment, prophylaxis and/or preventionof GH deficient growth disorder in a subject in need thereof.

In other aspects, the present disclosure provides polynucleotides thatencode the non-naturally occurring modified Cholix toxin-biologicallyactive cargo fusion molecules of the present disclosure; vectorscomprising polynucleotides encoding non-naturally occurring modifiedCholix toxin-biologically active cargo fusion molecules of thedisclosure; optionally, operably-linked to control sequences recognizedby a host cell transformed with the vector; host cells comprisingvectors comprising polynucleotides encoding non-naturally occurringmodified Cholix toxin-biologically active cargo fusion molecules of thedisclosure; a process for producing a non-naturally occurring modifiedCholix toxin-biologically active cargo fusion molecule of the disclosurecomprising culturing host cells comprising vectors comprisingpolynucleotides encoding non-naturally occurring modified Cholixtoxin-biologically active cargo fusion molecules of the disclosure suchthat the polynucleotide is expressed; and, optionally, recovering thenon-naturally occurring modified Cholix toxin-biologically active cargofusion molecule from the host cell culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overview of the strategy used for the C-terminalmodification of ntCholix to facilitate fusion with a cargo, in thisinstance, the cargo being Alexa488 flourescent dye.

FIG. 2 depicts the transport of ntCholix-Alexa488® across polarizedintestinal epithelial cells in vitro. Caco-2 cell monolayers wereexposed to test materials for 4 hr. The percentage of materialtransported was determined by standard curve analysis of fluorescencepresent in the samples and presented as an average (N=4). BSA-Alexa488was used as a control.

FIG. 3 depicts the genetic constructions of two exemplary Cholixtoxin-IL-10 fusion molecules evaluated herein. The N-terminus of a humanIL-10 monomer sequence was genetically attached to the C-terminus of amodified Cholix toxin (Cholix⁴¹⁵) using a stable non-cleavable linkersequence ((G₄S)₃) or a linker sequence that is a known substrate for thetobacco etch virus (TEV) protease. Each construct also contains anN-terminal Methionine (M).

FIG. 4 is a ribbon diagram representation of an exemplary “dimer Cholixtoxin-IL-10” fusion molecule after refolding that would be driven byIL-10 dimerization. The first 415 amino acids of Cholix toxin (SEQ IDNO: 1) are connected through a 16 amino acid linker (not shown) toconnect with the human IL-10 sequence. IL-10 dimerization is envisagedto result in purple Cholix⁴¹⁵/blue hIL-10 and orange Cholix⁴¹⁵/greenorganization shown.

FIG. 5 is a coomassie stained SDS PAGE of Cholix⁴¹⁵-TEV-IL-10 (depictedas “C”) and Cholix⁴¹⁵-(G₄S)₃-IL-10 (depicted as “N”) following inductionand expression from inclusion bodies. The expressed fusion moleculesdemonstrate the anticipated molecular size of ˜66 kDa that wascomparable to the calculated mass of 66380.78 and 65958.25 Daltons,respectively. SeeBlue® Plus2 Prestained MW standards are shown.

FIG. 6 is bar graph depicting the results of a flow cytometry assayusing a mouse macrophage-derived J774.2 cell line treated with anexemplary Cholix toxin-IL-10 fusion molecules of the present disclosureat two concentrations. % proliferation was measured at 48 hours posttreatment. Values represent n=4±standard deviation. The data shows that“dimer Cholix⁴¹⁵-(G₄S)₃-IL-10” fusion molecule demonstrates biologicallyactive IL-10.

FIG. 7 is a line graph depicting the results of an assay wherein thedimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule was tested for effects onthe barrier properties of Caco-2 cell monolayers in vitro.Fluorescein-labeled 70 kDa dextran and varying concentrations of dimerCholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule was added to the apical surfaceof these monolayers and the cumulative amount of florescence detected inthe basal compartment monitored over time by collecting 150 μL volumeswith replacement. Cumulative Basal Dextran levels (pmol) are plotted vstime. Each line represents the average (n=4) of basal fluorescencevalues measured at 0, 15, 30, 45, 60, 90, 120, 180, and 240 min.

FIG. 8 is a line graph depicting the results of an assay wherein thedimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule was tested for effects onthe barrier properties of Caco-2 cell monolayers in vitro.Fluorescein-labeled 70 kDa dextran and varying concentrations of dimerCholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule was added to the apical surfaceof these monolayers and the cumulative amount of florescence detected inthe basal compartment monitored over time.

FIGS. 9A and 9B are line graphs depicting the results an ELISA assayevaluating the ability of the dimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusionmolecule to move across Caco-2 cell monolayers. The cumulative amount ofdimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule reaching the basalcompartment over time following an apical addition at variousconcentrations denoted in the legend. Each line represents the average(n=4) of basal IL-10 levels measured at 0, 15, 30, 45, 60, 90, 120, 180,and 240 min. Cumulative IL-10 transported over time graphed over a rangeof 6A=8000 fmol IL-10 expanded and 6B=1000 fmol IL-10.

MODE(S) FOR CARRYING OUT THE INVENTION

As those in the art will appreciate, the foregoing description describescertain preferred embodiments of the invention in detail, and is thusonly representative and does not depict the actual scope of theinvention. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the invention defined by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The studies underlying the present invention relate to the use ofCholix-derived polypeptide sequences as the transporter domain to beused to prepare isolated delivery constructs to enhance intestinaldelivery of biologically-active therapeutics. Importantly, the systemsand methods described herein provide for the following: the ability todeliver macromolecule doses without injections; the ability to deliver“cargo” into intracellular compartments where their activity isrequired; and the delivery of nanoparticles and/or dendrimer-basedcarriers across biological membranes, which otherwise would have beenimpeded due to the barrier properties of most such membranes.

Mature Cholix toxin (“Cholix”) is an extremely active monomeric protein(molecular weight 70 kD) secreted by Vibrio cholerae, and which iscomposed of three prominent globular domains (Ia, II, and III) and onesmall subdomain (Ib) that connects domains II and III. The amino acidsequence of mature Cholix is provided in Jorgensen, R. et al., J BiolChem, 283(16):10671-10678 (2008) and references cited therein. TheCholix-derived polypeptide sequences used in the preparation of theisolated delivery constructs of the present invention will be derivedfrom the reported 634 amino acid protein sequence of mature Cholix.

Accordingly, the delivery constructs of the present invention comprise atransporter domain. A “transporter domain” as used herein refers tostructural domains which are capable of performing certain functions,e.g., cell recognition (i.e., comprise a receptor binding domain) andtranscytosis (i.e., comprise a transcytosis domain). Generally, thetransporter domains to be used in the preparation of the deliveryconstructs of the present invention are Cholix-derived polypeptidesequences that have structural domains corresponding to the functionaldomains, e.g., Ia and II, of Cholix.

In addition to the portions of the molecule that correspond to Cholixfunctional domains, the delivery constructs of this invention canfurther comprise a macromolecule for delivery to a biologicalcompartment of a subject. In certain embodiments, the macromolecule isselected from the group of a nucleic acid, a peptide, a polypeptide, aprotein, a polysaccharide, and a lipid. In further embodiments, thepolypeptide is selected from the group consisting of polypeptidehormones, cytokines, chemokines, growth factors, and clotting factorsthat are commonly administered to subjects by injection. The sequencesof all of these macromolecules are well known to those in the art, andattachment of these macromolecules to the delivery constructs is wellwithin the skill of those in the art using standard techniques.

The macromolecule can be introduced into any portion of the deliveryconstruct that does not disrupt a cell-binding or transcytosis activity.The macromolecule is connected to the remainder of the deliveryconstruct through a cleavable linker. “Linker” refers to a molecule thatjoins two other molecules, either covalently, or through ionic, van derWaals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizesto one complementary sequence at the 5′ end and to another complementarysequence at the 3′ end, thus joining two non-complementary sequences. A“cleavable linker” refers to a linker that can be degraded or otherwisesevered to separate the two components connected by the cleavablelinker. Cleavable linkers are generally cleaved by enzymes, typicallypeptidases, proteases, nucleases, lipases, and the like. Cleavablelinkers may also be cleaved by environmental cues, such as, for example,changes in temperature, pH, salt concentration, etc. when there is sucha change in environment following transcytosis of the delivery constructacross a polarized epithelial membrane.

In certain embodiments, the delivery constructs further comprise asecond macromolecule that is selected from the group consisting of anucleic acid, a peptide, a polypeptide, a protein, a lipid, and a smallorganic molecule and a second cleavable linker, wherein cleavage at saidsecond cleavable linker separates said second macromolecule from theremainder of said construct. In certain embodiments, the firstmacromolecule is a first polypeptide and said second macromolecule is asecond polypeptide. In certain embodiments, the first polypeptide andthe second polypeptide associate to form a multimer. In certainembodiments, the multimer is a dimer, tetramer, or octamer. In furtherembodiments, the dimer is an antibody.

In certain embodiments, the macromolecule can be selected to not becleavable by an enzyme present at the basal-lateral membrane of anepithelial cell. For example, the assays described in the examples canbe used to routinely test whether such a cleaving enzyme can cleave themacromolecule to be delivered. If so, the macromolecule can be routinelyaltered to eliminate the offending amino acid sequence recognized by thecleaving enzyme. The altered macromolecule can then be tested to ensurethat it retains activity using methods routine in the art.

In certain embodiments, the first and/or the second cleavable linker iscleavable by an enzyme that exhibits higher activity on thebasal-lateral side of a polarized epithelial cell than it does on theapical side of the polarized epithelial cell. In certain embodiments,the first and/or the second cleavable linker is cleavable by an enzymethat exhibits higher activity in the plasma than it does on the apicalside of a polarized epithelial cell.

In certain embodiments, the cleavable linker can be selected based onthe sequence, in the case of peptide, polypeptide, or proteinmacromolecules for delivery, to avoid the use of cleavable linkers thatcomprise sequences present in the macromolecule to be delivered. Forexample, if the macromolecule comprises AAL, the cleavable linker can beselected to be cleaved by an enzyme that does not recognize thissequence.

In addition to the portions of the molecule that correspond to Cholixfunctional domains, the delivery constructs of this invention canfurther comprise a “cargo” for delivery into intracellular compartmentswhere their activity is required. A “cargo” as used herein includes, butis not limited to: macromolecules, small molecules, siRNA, PNA, miRNA,DNA, plasmid and antisense molecules. Other examples of cargo that canbe delivered according to the present invention include, but are notlimited to, antineoplastic compounds, such as nitrosoureas, e.g.,carmustine, lomustine, semustine, strepzotocin; methylhydrazines, e.g.,procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids,estrogens, progestins, androgens, tetrahydrodesoxycaricosterone;immunoactive compounds such as immunosuppressives, e.g., pyrimethamine,trimethopterin, penicillamine, cyclosporine, azathioprine; andimmunostimulants, e.g., levamisole, diethyl dithiocarbamate,enkephalins, endorphins; antimicrobial compounds such as antibiotics,e.g., .beta.-lactam, penicillin, cephalosporins, carbapenims andmonobactams, .beta.-lactamase inhibitors, aminoglycosides, macrolides,tetracyclins, spectinomycin; antimalarials, amebicides; antiprotazoals;antifungals, e.g., amphotericin .beta., antivirals, e.g., acyclovir,idoxuridine, ribavirin, trifluridine, vidarbine, gancyclovir;parasiticides; antihalmintics; radiopharmaceutics; gastrointestinaldrugs; hematologic compounds; immunoglobulins; blood clotting proteins,e.g., antihemophilic factor, factor IX complex; anticoagulants, e.g.,dicumarol, heparin Na; fibrolysin inhibitors, e.g., tranexamic acid;cardiovascular drugs; peripheral anti-adrenergic drugs; centrally actingantihypertensive drugs, e.g., methyldopa, methyldopa HCl;antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl;drugs affecting renin-angiotensin system; peripheral vasodilators, e.g.,phentolamine; anti-anginal drugs; cardiac glycosides; inodilators, e.g.,amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;antidysrhythmics; calcium entry blockers; drugs affecting blood lipids,e.g., ranitidine, bosentan, rezulin; respiratory drugs; sypathomimeticdrugs, e.g., albuterol, bitolterol mesylate, dobutamine HCl, dopamineHCl, ephedrine So, epinephrine, fenfluramine HCl, isoproterenol HCl,methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrineHCl; cholinomimetic drugs, e.g., acetylcholine Cl; anticholinesterases,e.g., edrophonium Cl; cholinesterase reactivators; adrenergic blockingdrugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl,metoprolol, nadolol, phentolamine mesylate, propanolol HCl;antimuscarinic drugs, e.g., anisotropine methylbromide, atropineSO.sub.4, clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr;neuromuscular blocking drugs; depolarizing drugs, e.g., atracuriumbesylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl,tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g.,baclofen; neurotransmitters and neurotransmitter agents, e.g.,acetylcholine, adenosine, adenosine triphosphate; amino acidneurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenicamine neurotransmitters, e.g., dopamine, epinephrine, histamine,norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitricoxide, K.sup.+channel toxins; antiparkinson drugs, e.g., amaltidine HCl,benztropine mesylate, carbidopa; diuretic drugs, e.g., dichlorphenamide,methazolamide, bendroflumethiazide, polythiazide; antimigraine drugs,e.g, carboprost tromethamine mesylate, methysergide maleate.

The transporter domains of the delivery constructs of the presentinvention generally comprise a receptor binding domain. The receptorbinding domain can be any receptor binding domain known to one of skillin the art without limitation to bind to a cell surface receptor that ispresent on the apical membrane of an epithelial cell. Preferably, thereceptor binding domain binds specifically to the cell surface receptor.The receptor binding domain should bind to the cell surface receptorwith sufficient affinity to allow endocytosis of the delivery construct.

In certain embodiments, the receptor binding domain can comprise apeptide, a polypeptide, a protein, a lipid, a carbohydrate, or a smallorganic molecule, or a combination thereof. Examples of each of thesemolecules that bind to cell surface receptors present on the apicalmembrane of epithelial cells are well known to those of skill in theart. Suitable peptides or polypeptides include, but are not limited to,bacterial toxin receptor binding domains, such as the receptor bindingdomains from PE, cholera toxin, Cholix toxin, botulinum toxin, diptheriatoxin, shiga toxin, shiga-like toxin, etc.; antibodies, includingmonoclonal, polyclonal, and single-chain antibodies, or derivativesthereof, growth factors, such as EGF, IGF-I, IGF-II, IGF-III etc.;cytokines, such as IL-1, IL-2, IL-3, IL-6, etc; chemokines, such asMIP-1a, MIP-1b, MCAF, IL-8, etc.; and other ligands, such as CD4, celladhesion molecules from the immunoglobulin superfamily, integrins,ligands specific for the IgA receptor, etc. The skilled artisan canselect the appropriate receptor binding domain based upon the expressionpattern of the receptor to which the receptor binding domain binds.

The receptor binding domain can be attached to the remainder of thedelivery construct by any method or means known by one of skill in theart to be useful for attaching such molecules, without limitation. Incertain embodiments, the receptor binding domain is expressed togetherwith the remainder of the delivery construct as a fusion protein. Suchembodiments are particularly useful when the receptor binding domain andthe remainder of the construct are formed from peptides or polypeptides.

The transporter domains of the delivery constructs of the presentinvention further comprise a transcytosis domain. The transcytosisdomain can be any transcytosis domain known by one of skill in the artto effect transcytosis of chimeric proteins that have bound to a cellsurface receptor present on the apical membrane of an epithelial cell.In preferred embodiments, the transcytosis domain is domain II ofCholix.

Without intending to be limited to any particular theory or mechanism ofaction, the transcytosis domain is believed to permit the trafficking ofthe delivery construct through a polarized epithelial cell after theconstruct binds to a receptor present on the apical surface of thepolarized epithelial cell. Such trafficking through a polarizedepithelial cell is referred to herein as “transcytosis.” Thistrafficking permits the release of the delivery construct from thebasal-lateral membrane of the polarized epithelial cell.

For the delivery of cargo intended for intracellular activity, thedelivery construct comprises an endocytosis domain to traffic the cargointo the cell, and may also comprise a cleavable linker. This includesthe intracellular delivery of cargo using nanoparticle and/ordendrimer-based carriers targeted to the cell surface receptor bydecorating the carrier surface with one or more copies of theendocytosis domain, with or without the use of linkers.

Without intending to be limited to any particular theory or mechanism ofaction, the endocytosis domain is believed to permit the trafficking ofthe delivery construct into a cell after the construct binds to areceptor present on the surface of the cell. Such trafficking into acell is referred to herein as “endocytosis”. This trafficking permitsthe release of the delivery construct into the relevant intracellularcompartment, including (but not limited to) the nucleus and nuclearenvelope, ribosomal vesicles, endoplasmic reticulum, mitochondria, golgiapparatus, and cytosol.

In certain embodiments of the present invention, identification ofproteases and peptidases that function biological processes that occurat the basolateral surface of these cells will be evaluated. Theseproteases and peptidases would fall into several categories that can bedefined by their substrates: 1) pre-pro-hormones and enzymes that aresecreted from epithelial basolateral surfaces and required trimming fortheir activation, 2) active hormones or enzymes whose activity isneutralized by a cleavage event in order to regulate their activity, and3) systemic enzymes or growth factors whose actions at the basolateralsurface are altered by enzymatic modification. Examples of severalpotential activities that fall into these various categories and whichmight be of useful for the basolateral cleavage of carrier-linker-cargoconstructs include members of the S9B prolyl oligopeptidase subfamily,e.g., FAP and DDP IV, which have been described in the art.

The nucleic acid sequences and polynucleotides of the present inventioncan be prepared by any suitable method including, for example, cloningof appropriate sequences or by direct chemical synthesis by methods suchas the phosphotriester method of Narang, et al., Meth. Enzymol.,68:90-99 (1979); the phosphodiester method of Brown, et al., Meth.Enzymol., 68:109-151 (1979); the diethylphosphoramidite method ofBeaucage, et al., Tetra. Lett., 22:1859-1862 (1981); the solid phasephosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts., 22(20):1859-1862 (1981), e.g., using an automatedsynthesizer as described in, for example, Needham-VanDevanter, et al.Nucl. Acids Res. 12:6159-6168 (1984); and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

In a preferred embodiment, the nucleic acid sequences of this inventionare prepared by cloning techniques. Examples of appropriate cloning andsequencing techniques, and instructions sufficient to direct persons ofskill through many cloning exercises are found in Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold SpringHarbor Laboratory (1989)), Berger and Kimmel (eds.), GUIDE TO MOLECULARCLONING TECHNIQUES, Academic Press, Inc., San Diego Calif. (1987)), orAusubel, et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing and Wiley-Interscience, NY (1987). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAchemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Cells suitable for replicating and for supporting recombinant expressionof protein are well known in the art. Such cells may be transfected ortransduced as appropriate with the particular expression vector andlarge quantities of vector containing cells can be grown for seedinglarge scale fermenters to obtain sufficient quantities of the proteinfor clinical applications. Such cells may include prokaryoticmicroorganisms, such as E. coli; various eukaryotic cells, such asChinese hamster ovary cells (CHO), NSO, 292; Yeast; insect cells; andtransgenic animals and transgenic plants, and the like. Standardtechnologies are known in the art to express foreign genes in thesesystems.

The pharmaceutical compositions of the present invention comprise agenetic fusion or chemical construct of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Someexamples of pharmaceutically acceptable carriers are water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Additionalexamples of pharmaceutically acceptable substances are wetting agents orminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody. Except insofar as any conventionalexcipient, carrier or vehicle is incompatible with the deliveryconstructs of the present invention; its use in the pharmaceuticalpreparations of the invention is contemplated.

In certain embodiments, the pharmaceutical compositions of activecompounds may be prepared with a carrier that will protect thecomposition against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems (J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978).

In certain embodiments, the delivery constructs of the invention can beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) can also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the delivery constructs canbe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Generally, a pharmaceutically effective amount of the delivery constructof the invention is administered to a subject. The skilled artisan canreadily determine if the dosage of the delivery construct is sufficientto deliver an effective amount of the macromolecule, as described below.In certain embodiments, between about 1 .mu.g and about 1 g of deliveryconstruct is administered. In other embodiments, between about 10 .mu.gand about 500 mg of delivery construct is administered. In still otherembodiments, between about 10 .mu.g and about 100 mg of deliveryconstruct is administered. In yet other embodiments, between about 10.mu.g and about 1000 .mu.g of delivery construct is administered. Instill other embodiments, between about 10 .mu.g and about 250 .mu.g ofdelivery construct is administered. In yet other embodiments, betweenabout 10 .mu.g and about 100 .mu.g of delivery construct isadministered. Preferably, between about 10 .mu.g and about 50 .mu.g ofdelivery construct is administered.

The delivery constructs of the invention offer several advantages overconventional techniques for local or systemic delivery of macromoleculesto a subject. Foremost among such advantages is the ability to deliverthe macromolecule without using a needle to puncture the skin of thesubject. Many subjects require repeated, regular doses ofmacromolecules. For example, diabetics must inject insulin several timesper day to control blood sugar concentrations. Such subjects' quality oflife would be greatly improved if the delivery of a macromolecule couldbe accomplished without injection, by avoiding pain or potentialcomplications associated therewith.

Furthermore, many embodiments of the delivery constructs can beconstructed and expressed in recombinant systems. Recombinant technologyallows one to make a delivery construct having an insertion sitedesigned for introduction of any suitable macromolecule. Such insertionsites allow the skilled artisan to quickly and easily produce deliveryconstructs for delivery of new macromolecules, should the need to do soarise.

In addition, connection of the macromolecule to the remainder of thedelivery construct with a linker that is cleaved by an enzyme present ata basal-lateral membrane of an epithelial cell allows the macromoleculeto be liberated from the delivery construct and released from theremainder of the delivery construct soon after transcytosis across theepithelial membrane. Such liberation reduces the probability ofinduction of an immune response against the macromolecule. It alsoallows the macromolecule to interact with its target free from theremainder of the delivery construct.

Other advantages of the delivery constructs of the invention will beapparent to those of skill in the art.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those commonly used and well known in the art. The methodsand techniques of the present disclosure are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates (1992), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990), incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those commonly used and well known in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

Definitions

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Invarious embodiments, “peptides”, “polypeptides”, and “proteins” arechains of amino acids whose alpha carbons are linked through peptidebonds. The terminal amino acid at one end of the chain (amino terminal)therefore has a free amino group, while the terminal amino acid at theother end of the chain (carboxy terminal) has a free carboxyl group. Asused herein, the term “amino terminus” (abbreviated N-terminus) refersto the free α-amino group on an amino acid at the amino terminal of apeptide or to the α-amino group (imino group when participating in apeptide bond) of an amino acid at any other location within the peptide.Similarly, the term “carboxy terminus” refers to the free carboxyl groupon the carboxy terminus of a peptide or the carboxyl group of an aminoacid at any other location within the peptide. Peptides also includeessentially any polyamino acid including, but not limited to, peptidemimetics such as amino acids joined by an ether as opposed to an amidebond.

Polypeptides of the disclosure include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties. For example, single or multiple amino acidsubstitutions (e.g., conservative amino acid substitutions) may be madein the naturally occurring sequence (e.g., in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts). A“conservative amino acid substitution” refers to the substitution in apolypeptide of an amino acid with a functionally similar amino acid. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), and Threonine (T)

2) Aspartic acid (D) and Glutamic acid (E)

3) Asparagine (N) and Glutamine (Q)

4) Arginine (R) and Lysine (K)

5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

A “non-conservative amino acid substitution” refers to the substitutionof a member of one of these classes for a member from another class. Inmaking such changes, according to various embodiments, the hydropathicindex of amino acids may be considered. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in various embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In various embodiments,those that are within ±1 are included, and in various embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In various embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−.1);glutamate (+3.0.+−.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, in various embodiments, the substitutionof amino acids whose hydrophilicity values are within ±2 is included, invarious embodiments, those that are within ±1 are included, and invarious embodiments, those within ±0.5 are included.

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Residues ExemplarySubstitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys,Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn GluAsp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met,Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe LysArg, 1,4 Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu PheLeu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr SerSer Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe,Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. In variousembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in apolypeptide that correspond to amino acid residues important foractivity or structure in similar polypeptides. One skilled in the artmay opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of a polypeptide withrespect to its three-dimensional structure. In various embodiments, oneskilled in the art may choose to not make radical changes to amino acidresidues predicted to be on the surface of the polypeptide, since suchresidues may be involved in important interactions with other molecules.Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known to thoseskilled in the art. Such variants could be used to gather informationabout suitable variants. For example, if one discovered that a change toa particular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change can beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

The term “polypeptide fragment” and “truncated polypeptide” as usedherein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion as compared to a corresponding full-lengthprotein. In various embodiments, fragments can be, e.g., at least 5, atleast 10, at least 25, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least450, at least 500, at least 600, at least 700, at least 800, at least900 or at least 1000 amino acids in length. In various embodiments,fragments can also be, e.g., at most 1000, at most 900, at most 800, atmost 700, at most 600, at most 500, at most 450, at most 400, at most350, at most 300, at most 250, at most 200, at most 150, at most 100, atmost 50, at most 25, at most 10, or at most 5 amino acids in length. Afragment can further comprise, at either or both of its ends, one ormore additional amino acids, for example, a sequence of amino acids froma different naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence).

The terms “polypeptide variant” and “polypeptide mutant” as used hereinrefers to a polypeptide that comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. In various embodiments, the number of amino acid residues tobe inserted, deleted, or substituted can be, e.g., at least 1, at least2, at least 3, at least 4, at least 5, at least 10, at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 350, at least 400, at least 450 or at least 500amino acids in length. Variants of the present disclosure include fusionproteins.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified, e.g., conjugation to another chemical moiety suchas, for example, polyethylene glycol, albumin (e.g., human serumalbumin), phosphorylation, and glycosylation.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. In variousembodiments, the % identity is selected from, e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% or more sequence identity to agiven sequence. In various embodiments, the % identity is in the rangeof, e.g., about 60% to about 70%, about 70% to about 80%, about 80% toabout 85%, about 85% to about 90%, about 90% to about 95%, or about 95%to about 99%.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. In various embodiments, the % homology is selectedfrom, e.g., at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ormore sequence homology to a given sequence. In various embodiments, the% homology is in the range of, e.g., about 60% to about 70%, about 70%to about 80%, about 80% to about 85%, about 85% to about 90%, about 90%to about 95%, or about 95% to about 99%.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.USA, 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is, e.g., at most 0.1, at most 0.01, or at most 0.001.

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is substantiallyidentical to the nucleotide sequence of the polynucleotide bindingpartner of the second polynucleotide, or if the first polynucleotide canhybridize to the second polynucleotide under stringent hybridizationconditions.

“Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence-dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids can be found in Tijssen, 1993, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, N.Y.; Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 3.sup.rd ed., NY; and Ausubel et al., eds., Current Edition,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY.

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe. Anexample of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than about 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes. See Sambrook et al. for adescription of SSC buffer. A high stringency wash can be preceded by alow stringency wash to remove background probe signal. An exemplarymedium stringency wash for a duplex of, e.g., more than about 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An exemplary lowstringency wash for a duplex of, e.g., more than about 100 nucleotides,is 4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties. In instances where a probe provides a point ofinitiation for synthesis of a complementary polynucleotide, a probe canalso be a primer.

A “vector” is a polynucleotide that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A “regulatory sequence” is a nucleic acid that affects the expression(e.g., the level, timing, or location of expression) of a nucleic acidto which it is operably linked. The regulatory sequence can, forexample, exert its effects directly on the regulated nucleic acid, orthrough the action of one or more other molecules (e.g., polypeptidesthat bind to the regulatory sequence and/or the nucleic acid). Examplesof regulatory sequences include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Furtherexamples of regulatory sequences are described in, for example, Goeddel,1990, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res.23:3605-06. A nucleotide sequence is “operably linked” to a regulatorysequence if the regulatory sequence affects the expression (e.g., thelevel, timing, or location of expression) of the nucleotide sequence.

A “host cell” is a cell that can be used to express a polynucleotide ofthe disclosure. A host cell can be a prokaryote, for example, E. coli,or it can be a eukaryote, for example, a single-celled eukaryote (e.g.,a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plantcell), an animal cell (e.g., a human cell, a monkey cell, a hamstercell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.Typically, a host cell is a cultured cell that can be transformed ortransfected with a polypeptide-encoding nucleic acid, which can then beexpressed in the host cell. The phrase “recombinant host cell” can beused to denote a host cell that has been transformed or transfected witha nucleic acid to be expressed. A host cell also can be a cell thatcomprises the nucleic acid but does not express it at a desired levelunless a regulatory sequence is introduced into the host cell such thatit becomes operably linked with the nucleic acid. It is understood thatthe term host cell refers not only to the particular subject cell but tothe progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “isolated molecule” (where the molecule is, for example, apolypeptide or a polynucleotide) is a molecule that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) issubstantially free of other molecules from the same species (3) isexpressed by a cell from a different species, or (4) does not occur innature. Thus, a molecule that is chemically synthesized, or expressed ina cellular system different from the cell from which it naturallyoriginates, will be “isolated” from its naturally associated components.A molecule also may be rendered substantially free of naturallyassociated components by isolation, using purification techniques wellknown in the art. Molecule purity or homogeneity may be assayed by anumber of means well known in the art. For example, the purity of apolypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60% to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and e.g., will be over 99%pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences. A “cleavable linker” refers toa linker that can be degraded or otherwise severed to separate the twocomponents connected by the cleavable linker. Cleavable linkers aregenerally cleaved by enzymes, typically peptidases, proteases,nucleases, lipases, and the like. Cleavable linkers may also be cleavedby environmental cues, such as, for example, specific enzymaticactivities, changes in temperature, pH, salt concentration, etc. whenthere is such a change in environment following transcytosis of thefusion molecules across a polarized epithelial membrane.

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in an animal. A pharmaceutical composition comprisesa pharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result

“Pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers, vehicles, buffers, and excipients, such as aphosphate buffered saline solution, 5% aqueous solution of dextrose, andemulsions, such as an oil/water or water/oil emulsion, and various typesof wetting agents and/or adjuvants. Suitable pharmaceutical carriers andformulations are described in Remington's Pharmaceutical Sciences, 21stEd. 2005, Mack Publishing Co, Easton. A “pharmaceutically acceptablesalt” is a salt that can be formulated into a compound forpharmaceutical use including, e.g., metal salts (sodium, potassium,magnesium, calcium, etc.) and salts of ammonia or organic amines.

The terms “treat”, “treating” and “treatment” refer to a method ofalleviating or abrogating a biological disorder and/or at least one ofits attendant symptoms. As used herein, to “alleviate” a disease,disorder or condition means reducing the severity and/or occurrencefrequency of the symptoms of the disease, disorder, or condition.Further, references herein to “treatment” include references tocurative, palliative and prophylactic treatment.

Modified Cholix Toxin Polypeptides

Mature Cholix toxin (Jorgensen, R. et al., J Biol Chem283(16):10671-10678 (2008)) as used herein is a 70.7 kD, 634 residueprotein, whose sequence is set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLIPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK

In various embodiments, the Cholix toxin has an amino acid sequence thatshares an observed homology of, e.g., at least about 75%, at least about80%, at least about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least about 99% with the sequence of SEQ ID NO: 1.

An exemplary nucleic acid encoding the mature Cholix toxin is set forthin SEQ ID NO: 2:

(SEQ ID NO: 2) ATGGTCGAAGAAGCTTTAAACATCTTTGATGAATGCCGTTCGCCATGTTCGTTGACCCCGGAACCGGGTAAGCCGATTCAATCAAAACTGTCTATCCCTAGTGATGTTGTTCTGGATGAAGGTGTTCTGTATTACTCGATGACGATTAATGATGAGCAGAATGATATTAAGGATGAGGACAAAGGCGAGTCCATTATCACTATTGGTGAATTTGCCACAGTACGCGCGACTAGACATTATGTTAATCAAGATGCGCCTTTTGGTGTCATCCATTTAGATATTACGACAGAAAATGGTACAAAAACGTACTCTTATAACCGCAAAGAGGGTGAATTTGCAATCAATTGGTTAGTGCCTATTGGTGAAGATTCTCCTGCAAGCATCAAAATCTCCGTTGATGAGCTCGATCAGCAACGCAATATCATCGAGGTGCCTAAACTGTATAGTATTGATCTCGATAACCAAACGTTAGAGCAGTGGAAAACCCAAGGTAATGTTTCTTTTTCGGTAACGCGTCCTGAACATAATATCGCTATCTCTTGGCCAAGCGTGAGTTACAAAGCAGCGCAGAAAGAGGGTTCACGCCATAAGCGTTGGGCTCATTGGCATACAGGCTTAGCACTGTGTTGGCTTGTGCCAATGGATGCTATCTATAACTATATCACCCAGCAAAATTGTACTTTAGGGGATAATTGGTTTGGTGGCTCTTATGAGACTGTTGCAGGCACTCCGAAGGTGATTACGGTTAAGCAAGGGATTGAACAAAAGCCAGTTGAGCAGCGCATCCATTTCTCCAAGGGGAATGCGATGAGCGCACTTGCTGCTCATCGCGTCTGTGGTGTGCCATTAGAAACTTTGGCGCGCAGTCGCAAACCTCGTGATCTGACGGATGATTTATCATGTGCCTATCAAGCGCAGAATATCGTGAGTTTATTTGTCGCGACGCGTATCCTGTTCTCTCATCTGGATAGCGTATTTACTCTGAATCTTGACGAACAAGAACCAGAGGTGGCTGAACGTCTAAGTGATCTTCGCCGTATCAATGAAAATAACCCGGGCATGGTTACACAGGTTTTAACCGTTGCTCGTCAGATCTATAACGATTATGTCACTCACCATCCGGGCTTAACTCCTGAGCAAACCAGTGCGGGTGCACAAGCTGCCGATATCCTCTCTTTATTTTGCCCAGATGCTGATAAGTCTTGTGTGGCTTCAAACAACGATCAAGCCAATATCAACATCGAGTCTCGTTCTGGCCGTTCATATTTGCCTGAAAACCGTGCGGTAATCACCCCTCAAGGCGTCACAAATTGGACTTACCAGGAACTCGAAGCAACACATCAAGCTCTGACTCGTGAGGGTTATGTGTTCGTGGGTTACCATGGTACGAATCATGTCGCTGCGCAAACCATCGTGAATCGCATTGCCCCTGTTCCGCGCGGCAACAACACTGAAAACGAGGAAAAGTGGGGCGGGTTATATGTTGCAACTCACGCTGAAGTTGCCCATGGTTATGCTCGCATCAAAGAAGGGACAGGGGAGTATGGCCTTCCGACCCGTGCTGAGCGCGACGCTCGTGGGGTAATGCTGCGCGTGTATATCCCTCGTGCTTCATTAGAACGTTTTTATCGCACGAATACACCTTTGGAAAATGCTGAGGAGCATATCACGCAAGTGATTGGTCATTCTTTGCCATTACGCAATGAAGCATTTACTGGTCCAGAAAGTGCGGGCGGGGAAGACGAAACTGTCATTGGCTGGGATATGGCGATTCATGCAGTTGCGATCCCTTCGACTATCCCAGGGAACGCTTACGAAGAATTGGCGATTGATGAGGAGGCTGTTGCAAAAGAGCAATCGATTAGCACAAAACCACCTTATAAAGAGCGCAAAGATGAAC TTAAG

In various embodiments, the Cholix toxin contains an nucleic acidsequence that shares an observed homology of, e.g., at least about 75%,at least about 80%, at least about 85%, at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with the sequence ofSEQ ID NO: 2.

In various embodiments, the modified Cholix toxin used in thepreparation of the fusion molecules is a truncated Cholix toxin, whereinthe fusion molecule has the ability to activate the receptor for thebiologically active cargo. A truncated Cholix toxin as described hereinwill be identified by reference to the amino acid residues comprisingthe truncated Cholix toxin, e.g., a truncated Cholix toxin consisting ofamino acid residues 1-386 of SEQ ID NO: 1 will be identified asCholix³⁸⁶.

In various embodiments, the modified Cholix toxin used in thepreparation of the fusion molecule is mutated Cholix toxin. As describedherein, a mutated Cholix toxin wherein the mutation involves an aminoacid residue deletion will be identified by reference to the amino acidresidue being deleted, e.g., a mutated Cholix toxin wherein amino acidE581 of SEQ ID NO: 1 has been deleted, the will be identified as “CholixΔE581”. A mutated Cholix toxin wherein the mutation involves an aminoacid residue substitution will be identified by reference to theparticular amino acid substitution at a specific amino acid residue.Thus, e.g., the term “S30A” indicates that the “S” (serine, in standardsingle letter code) residue at position 30 in SEQ ID NO: 1 has beensubstituted with an “A” (alanine, in standard single letter code) evenif the residue appears in a truncated Cholix toxin, and the modifiedtoxin will be identified as “Cholix^(S30A)”.

Cholix toxin Domain Ia (amino acids 1-265 of SEQ ID NO: 1) is a“receptor binding domain” that functions as a ligand for a cell surfacereceptor and mediates binding of the fusion molecule to a cell, e.g.,Domain Ia will bind to a cell surface receptor that is present on theapical membrane of an epithelial cell, with sufficient affinity to allowendocytosis of the fusion molecule. Domain 1a can bind to any receptorknown to be present on the apical membrane of an epithelial cell by oneof skill in the art without limitation. For example, the receptorbinding domain can bind to α2-MR. Conservative or nonconservativesubstitutions can be made to the amino acid sequence of domain Ia, aslong as the ability to mediate binding of the fusion molecule to a cellis not substantially eliminated. In various embodiments, the fusionmolecules comprise a Cholix toxin comprising a mutated domain Ia.

In various embodiments, domain Ia comprises an antigen presenting cell(APC) receptor binding domain. In various embodiments, the APC receptorbinding domain is the cell recognition domain of Cholix domain Ia or aportion of Cholix domain Ia sufficient to engage with a cell surfacereceptor on APCs.

In various embodiments, the APC receptor binding domain binds to areceptor identified as present on a dendritic cell or other APC.Examples of cell surface receptors on APCs can include, but are notlimited to, DEC-205 (CD205), CD207, CD209, CD11a, CD11b, CD11c, CD36,CD14, CD50, CD54, CD58, CD68, CD80, CD83, CD86, CD102, CD3, CD14, CD19,Clec9a, CMFR-44, dectin-1,dectin-2, FLT3, HLA-DR, LOX-1, MHC II, BDCA-1,DC-SIGN, Toll-like receptors (TLR)-2, -3, -4, and -7, anda2-macroglobulin receptor (“α2-MR”). In various embodiments, the APCreceptor binding domain is α2-MR.

Cholix toxin Domain II (amino acids 266-386 of SEQ ID NO: 1) is a“transcytosis domain” that mediates transcytosis from a lumen borderingthe apical surface of a mucous membrane to the basolateral side of amucous membrane. As referred to herein, “transcytosis” refers to thetrafficking of the fusion molecule through a polarized epithelial cell.Such trafficking permits the release of the biologically active cargofrom the basolateral membrane of the polarized epithelial cell. Thefusion molecules of the present disclosure may comprise a modifiedCholix toxin comprising the entire amino acid sequence of Domain II, ormay comprise portions of Domain II, so long as transcytosis acitivity isnot substantially eliminated. Further, conservative or nonconservativesubstitutions can be made to the amino acid sequence of the transcytosisdomain, as long as transcytosis activity is not substantiallyeliminated. A representative assay that can routinely be used by one ofskill in the art to determine whether a transcytosis domain hastranscytosis activity is described herein. As used herein, thetranscytosis activity is not substantially eliminated so long as theactivity is, e.g., at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% as compared to a modified Cholix toxin comprisingthe entire amino acid sequence of Domain II.

In various embodiments, the non-naturally occurring fusion moleculescomprise a modified Cholix toxin truncated at an amino acid residuewithin Cholix toxin domain II, wherein the fusion molecule has theability to activate the receptor for the biologically active cargo. Inone embodiment, the truncated Cholix toxin is Cholix³⁸⁶ (SEQ ID NO: 3).In one embodiment, the truncated Cholix toxin is Cholix³⁸⁵ (SEQ ID NO:4). In one embodiment, the truncated Cholix toxin is Cholix³⁸⁴ (SEQ IDNO: 5). In one embodiment, the truncated Cholix toxin is Cholix³⁸³ (SEQID NO: 6). In one embodiment, the truncated Cholix toxin is Cholix³⁸²(SEQ ID NO: 7). In one embodiment, the truncated Cholix toxin isCholix³⁸¹ (SEQ ID NO: 8). In one embodiment, the truncated Cholix toxinis Cholix³⁸⁰ (SEQ ID NO: 9). In one embodiment, the truncated Cholixtoxin is Cholix³⁷⁹ (SEQ ID NO: 10). In one embodiment, the truncatedCholix toxin is Cholix³⁷⁸ (SEQ ID NO: 11). In one embodiment, thetruncated Cholix toxin is Cholix³⁷⁷ (SEQ ID NO: 12). In one embodiment,the truncated Cholix toxin is Cholix³⁷⁶ (SEQ ID NO: 13). In oneembodiment, the truncated Cholix toxin is Cholix³⁷⁵ (SEQ ID NO: 14). Inone embodiment, the truncated Cholix toxin is Cholix³⁷⁴ (SEQ ID NO: 15).In one embodiment, the truncated Cholix toxin is Cholix³⁷³ (SEQ ID NO:16). In one embodiment, the truncated Cholix toxin is Cholix³⁷² (SEQ IDNO: 17). In one embodiment, the truncated Cholix toxin is Cholix³⁷¹ (SEQID NO: 18). In one embodiment, the truncated Cholix toxin is Cholix³⁷⁰(SEQ ID NO: 19). In one embodiment, the truncated Cholix toxin isCholix³⁶⁹ (SEQ ID NO: 20). In one embodiment, the truncated Cholix toxinis Cholix³⁶⁸ (SEQ ID NO: 21). In one embodiment, the truncated Cholixtoxin is Cholix³⁶⁷ (SEQ ID NO: 22). In one embodiment, the truncatedCholix toxin is Cholix³⁶⁶ (SEQ ID NO: 23). In one embodiment, thetruncated Cholix toxin is Cholix³⁶⁵ (SEQ ID NO: 24). In one embodiment,the truncated Cholix toxin is Cholix³⁶⁴ (SEQ ID NO: 25). In oneembodiment, the truncated Cholix toxin is Cholix³⁶³ (SEQ ID NO: 26). Inone embodiment, the truncated Cholix toxin is Cholix³⁶² (SEQ ID NO: 27).In one embodiment, the truncated Cholix toxin is Cholix³⁶¹ (SEQ ID NO:28). In one embodiment, the truncated Cholix toxin is Cholix³⁶⁰ (SEQ IDNO: 29). In one embodiment, the truncated Cholix toxin is Cholix³⁵⁹ (SEQID NO: 30). In one embodiment, the truncated Cholix toxin is Cholix³⁵⁸(SEQ ID NO: 31). In one embodiment, the truncated Cholix toxin isCholix³⁵⁷ (SEQ ID NO: 32). In one embodiment, the truncated Cholix toxinis Cholix³⁵⁶ (SEQ ID NO: 33). In one embodiment, the truncated Cholixtoxin is Cholix³⁵⁵ (SEQ ID NO: 34). In one embodiment, the truncatedCholix toxin is Cholix³⁵⁴ (SEQ ID NO: 35). In one embodiment, thetruncated Cholix toxin is Cholix³⁵³ (SEQ ID NO: 36). In one embodiment,the truncated Cholix toxin is Cholix³⁵² (SEQ ID NO: 37). In oneembodiment, the truncated Cholix toxin is Cholix³⁵¹ (SEQ ID NO: 38). Inone embodiment, the truncated Cholix toxin is Cholix³⁵⁰ (SEQ ID NO: 39).In one embodiment, the truncated Cholix toxin is Cholix³⁴⁹ (SEQ ID NO:40). In one embodiment, the truncated Cholix toxin is Cholix³⁴⁸ (SEQ IDNO: 41).

Cholix toxin Domain Ib (amino acids 387-425 of SEQ ID NO: 1) is notessential for any known activity of Cholix, including cell binding,translocation, ER retention or ADP ribosylation activity. In variousembodiments, the non-naturally occurring fusion molecules comprise amodified Cholix toxin truncated at an amino acid residue within Cholixtoxin domain Ib, wherein the fusion molecule has the ability to activatethe receptor for the biologically active cargo. In one embodiment, thetruncated Cholix toxin is Cholix⁴²⁵ (SEQ ID NO: 42). In one embodiment,the truncated Cholix toxin is Cholix⁴²⁴ (SEQ ID NO: 43). In oneembodiment, the truncated Cholix toxin is Cholix⁴²³ (SEQ ID NO: 44). Inone embodiment, the truncated Cholix toxin is Cholix⁴²² (SEQ ID NO: 45).In one embodiment, the truncated Cholix toxin is Cholix⁴²¹ (SEQ ID NO:46). In one embodiment, the truncated Cholix toxin is Cholix⁴²⁰ (SEQ IDNO: 47). In one embodiment, the truncated Cholix toxin is Cholix⁴¹⁹ (SEQID NO: 48). In one embodiment, the truncated Cholix toxin is Cholix⁴¹⁸(SEQ ID NO: 49). In one embodiment, the truncated Cholix toxin isCholix⁴¹⁷ (SEQ ID NO: 50). In one embodiment, the truncated Cholix toxinis Cholix⁴¹⁶ (SEQ ID NO: 51). In one embodiment, the truncated Cholixtoxin is Cholix⁴¹⁵ (SEQ ID NO: 52). In one embodiment, the truncatedCholix toxin is Cholix⁴¹⁴ (SEQ ID NO: 53). In one embodiment, thetruncated Cholix toxin is Cholix⁴¹³ (SEQ ID NO: 54). In one embodiment,the truncated Cholix toxin is Cholix⁴¹² (SEQ ID NO: 55). In oneembodiment, the truncated Cholix toxin is Cholix⁴¹¹ (SEQ ID NO: 56). Inone embodiment, the truncated Cholix toxin is Cholix⁴¹⁰ (SEQ ID NO: 57).In one embodiment, the truncated Cholix toxin is Cholix⁴⁰⁹ (SEQ ID NO:58). In one embodiment, the truncated Cholix toxin is Cholix⁴⁰⁸ (SEQ IDNO: 59). In one embodiment, the truncated Cholix toxin is Cholix⁴⁰⁷ (SEQID NO: 60). In one embodiment, the truncated Cholix toxin is Cholix⁴⁰⁶(SEQ ID NO: 61). In one embodiment, the truncated Cholix toxin isCholix⁴⁰⁵ (SEQ ID NO: 62). In one embodiment, the truncated Cholix toxinis Cholix⁴⁰⁴ (SEQ ID NO: 63). In one embodiment, the truncated Cholixtoxin is Cholix⁴⁰³ (SEQ ID NO: 64). In one embodiment, the truncatedCholix toxin is Cholix⁴⁰² (SEQ ID NO: 65). In one embodiment, thetruncated Cholix toxin is Cholix⁴⁰¹ (SEQ ID NO: 66). In one embodiment,the truncated Cholix toxin is Cholix⁴⁰⁰ (SEQ ID NO: 67). In oneembodiment, the truncated Cholix toxin is Cholix³⁹⁹ (SEQ ID NO: 68). Inone embodiment, the truncated Cholix toxin is Cholix³⁹⁸ (SEQ ID NO: 69).In one embodiment, the truncated Cholix toxin is Cholix³⁹⁷ (SEQ ID NO:70). In one embodiment, the truncated Cholix toxin is Cholix³⁹⁶ (SEQ IDNO: 71). In one embodiment, the truncated Cholix toxin is Cholix³⁹⁵ (SEQID NO: 72). In one embodiment, the truncated Cholix toxin is Cholix³⁹⁴(SEQ ID NO: 73). In one embodiment, the truncated Cholix toxin isCholix³⁹³ (SEQ ID NO: 74). In one embodiment, the truncated Cholix toxinis Cholix³⁹² (SEQ ID NO: 75). In one embodiment, the truncated Cholixtoxin is Cholix³⁹¹ (SEQ ID NO: 76). In one embodiment, the truncatedCholix toxin is Cholix³⁹⁰ (SEQ ID NO: 77). In one embodiment, thetruncated Cholix toxin is Cholix³⁸⁹ (SEQ ID NO: 78). In one embodiment,the truncated Cholix toxin is Cholix³⁸⁸ (SEQ ID NO: 79). In oneembodiment, the truncated Cholix toxin is Cholix³⁸⁷ (SEQ ID NO: 80).

Cholix toxin Domain III (amino acids 426-634 of SEQ ID NO: 1) isresponsible for cytotoxicity and includes an endoplasmic reticulumretention sequence. Domain III mediates ADP ribosylation of elongationfactor 2 (“EF2”), which inactivates protein synthesis. A Cholix that“lacks endogenous ADP ribosylation activity” or a “detoxified Cholix”refers to any Cholix described herein (including modified variants) thatdoes not comprise Cholix domain III or which has been modified withindomain III in a manner which detoxifies the molecule. For example,deletion of the glutamic acid (Glu) residue at amino acid position 581of SEQ ID NO: 1 detoxifies the molecule. This detoxified Cholix isreferred to as “Cholix ΔE581”. In various embodiments, the portion ofCholix domain III other than the ER retention signal can be replaced byanother amino acid sequence. This amino acid sequence can itself benon-immunogenic, slightly immunogenic, or highly immunogenic. A highlyimmunogenic ER retention domain is preferable for use in eliciting ahumoral immune response. For example, Cholix domain III is itself highlyimmunogenic and can be used in fusion molecules where a robust humoralimmune response is desired.

As used herein, “a detoxified Cholix sequence” may be a full lengthsequence or portion(s) of the full length sequence. Generally, adetoxified Cholix sequence has one or more domains or portions ofdomains with certain biological activities of a detoxified Cholix, suchas a cell recognition domain, a translocation domain, or an endoplasmicreticulum retention domain. For example, a detoxified Cholix sequencemay include only domain II and detoxified domain III. In anotherexample, a detoxified Cholix sequence may include only domain Ia, domainII, and detoxified domain III. In another example, a detoxified Cholixsequence may include all of domains Ia, Ib, II, and detoxified III.Therefore, a detoxified Cholix sequence may be a contiguous sequence ofthe native Cholix, or it can be a sequence comprised of non-contiguoussubsequences of the native Cholix that lacks ADP ribosylation activity.In one embodiment of the present disclosure, the non-naturally occurringfusion molecule comprises a mutated modified Cholix toxin, designatedherein as Cholix toxin ΔE581, having the amino acid sequence set forthin SEQ ID NO: 81.

Biologically Active Cargo

In addition to the modified Cholix toxin polypeptide, the fusionmolecules of the present disclosure further comprise a biologicallyactive cargo for delivery to a subject. A “biologically active cargo” asused herein includes, but is not limited to: a macromolecule, smallmolecule, peptide, polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA,antisense molecule, antibody, DNA, plasmid, vaccine, polymernanoparticle, or catalytically-active material.

In various embodiments, the biologically active cargo is a macromoleculethat can perform a desirable biological activity when introduced to thebloodstream of the subject. For example, the biologically active cargocan have receptor binding activity, enzymatic activity, messengeractivity (i.e., act as a hormone, cytokine, neurotransmitter, or othersignaling molecule), luminescent or other detectable activity, orregulatory activity, or any combination thereof. In certain diagnosticembodiments, the biologically active cargo can be conjugated to or canitself be a pharmaceutically acceptable gamma-emitting moiety, includingbut not limited to, indium and technetium, magnetic particles,radiopaque materials such as air or barium and fluorescent compounds.

In various embodiments, the biologically active cargo of the fusionmolecule can exert its effects in biological compartments of the subjectother than the subject's blood. For example, in various embodiments, thebiologically active cargo can exert its effects in the lymphatic system.In other embodiments, the biologically active cargo can exert itseffects in an organ or tissue, such as, for example, the subject'sliver, heart, lungs, pancreas, kidney, brain, bone marrow, etc. In suchembodiments, the biologically active cargo may or may not be present inthe blood, lymph, or other biological fluid at detectableconcentrations, yet may still accumulate at sufficient concentrations atits site of action to exert a biological effect.

In various embodiments, the biologically active cargo is a protein thatcomprises more than one polypeptide subunit. For example, the proteincan be a dimer, trimer, or higher order multimer. In variousembodiments, two or more subunits of the protein can be connected with acovalent bond, such as, for example, a disulfide bond. In otherembodiments, the subunits of the protein can be held together withnon-covalent interactions. One of skill in the art can routinelyidentify such proteins and determine whether the subunits are properlyassociated using, for example, an immunoassay.

In various embodiments, the biologically active cargo to be delivered isselected from, e.g., cytokines and cytokine receptors such asInterleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, lymphokine inhibitory factor, macrophage colony stimulatingfactor, platelet derived growth factor, stem cell factor, tumor growthfactor-β, tumor necrosis factor, lymphotoxin, Fas, granulocyte colonystimulating factor, granulocyte macrophage colony stimulating factor,interferon-α, interferon-β, interferon-γ, growth factors and proteinhormones such as erythropoietin, angiogenin, hepatocyte growth factor,fibroblast growth factor, keratinocyte growth factor, nerve growthfactor, tumor growth factor-α, thrombopoietin, thyroid stimulatingfactor, thyroid releasing hormone, neurotrophin, epidermal growthfactor, VEGF, ciliary neurotrophic factor, LDL, somatomedin, insulingrowth factor, insulin-like growth factor I and II, chemokines such asENA-78, ELC, GRO-α, GROβ, GRO-γ, HRG, LEF, IP-10, MCP-1, MCP-2, MCP-3,MCP-4, MIP-1-α, MIP-1-β, MG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES,lymphotactin, eotaxin-1, eotaxin-2, TARO, TECK, WAP-1, WAP-2, GCP-1,GCP-2; α-chemokine receptors, e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, CXCR7; and β-chemokine receptors, e.g., CCR1, CCR2, CCR3, CCR4,CCR5, CCR6, CCR7.

Other examples of biologically active cargo that can be deliveredaccording to the present disclosure include, but are not limited to,antineoplastic compounds, such as nitrosoureas, e.g., carmustine,lomustine, semustine, strepzotocin; methylhydrazines, e.g.,procarbazine, dacarbazine; steroid hormones, e.g., glucocorticoids,estrogens, progestins, androgens, tetrahydrodesoxycaricosterone;immunoactive compounds such as immunosuppressives, e.g., pyrimethamine,trimethopterin, penicillamine, cyclosporine, azathioprine; andimmunostimulants, e.g., levamisole, diethyl dithiocarbamate,enkephalins, endorphins; antimicrobial compounds such as antibiotics,e.g., β-lactam, penicillin, cephalosporins, carbapenims and monobactams,β-lactamase inhibitors, aminoglycosides, macrolides, tetracyclins,spectinomycin; antimalarials, amebicides; antiprotazoals; antifungals,e.g., amphotericin β, antivirals, e.g., acyclovir, idoxuridine,ribavirin, trifluridine, vidarbine, gancyclovir; parasiticides;antihalmintics; radiopharmaceutics; gastrointestinal drugs; hematologiccompounds; immunoglobulins; blood clotting proteins, e.g.,antihemophilic factor, factor IX complex; anticoagulants, e.g.,dicumarol, heparin Na; fibrolysin inhibitors, e.g., tranexamic acid;cardiovascular drugs; peripheral anti-adrenergic drugs; centrally actingantihypertensive drugs, e.g., methyldopa, methyldopa HCl;antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl;drugs affecting renin-angiotensin system; peripheral vasodilators, e.g.,phentolamine; anti-anginal drugs; cardiac glycosides; inodilators, e.g.,amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;antidysrhythmics; calcium entry blockers; drugs affecting blood lipids,e.g., ranitidine, bosentan, rezulin; respiratory drugs; sypathomimeticdrugs, e.g., albuterol, bitolterol mesylate, dobutamine HCl, dopamineHCl, ephedrine So, epinephrine, fenfluramine HCl, isoproterenol HCl,methoxamine HCl, norepinephrine bitartrate, phenylephrine HCl, ritodrineHCl; cholinomimetic drugs, e.g., acetylcholine Cl; anticholinesterases,e.g., edrophonium Cl; cholinesterase reactivators; adrenergic blockingdrugs, e.g., acebutolol HCl, atenolol, esmolol HCl, labetalol HCl,metoprolol, nadolol, phentolamine mesylate, propanolol HCl;antimuscarinic drugs, e.g., anisotropine methylbromide, atropine SO₄,clinidium Br, glycopyrrolate, ipratropium Br, scopolamine HBr;neuromuscular blocking drugs; depolarizing drugs, e.g., atracuriumbesylate, hexafluorenium Br, metocurine iodide, succinylcholine Cl,tubocurarine Cl, vecuronium Br; centrally acting muscle relaxants, e.g.,baclofen; neurotransmitters and neurotransmitter agents, e.g.,acetylcholine, adenosine, adenosine triphosphate; amino acidneurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenicamine neurotransmitters, e.g., dopamine, epinephrine, histamine,norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitricoxide, K⁺ channel toxins; antiparkinson drugs, e.g., amaltidine HCl,benztropine mesylate, carbidopa; diuretic drugs, e.g., dichlorphenamide,methazolamide, bendroflumethiazide, polythiazide; antimigraine drugs,e.g, carboprost tromethamine mesylate, methysergide maleate.

Still other examples of biologically active cargo that can be deliveredaccording to the present disclosure include, but are not limited to,hormones such as pituitary hormones, e.g., chorionic gonadotropin,cosyntropin, menotropins, somatotropin, iorticotropin, protirelin,thyrotropin, vasopressin, lypressin; adrenal hormones, e.g.,beclomethasone dipropionate, betamethasone, dexarnethasone,triamcinolone; pancreatic hormones, e.g., glucagon, insulin; parathyroidhormone, e.g., dihydrochysterol; thyroid hormones, e.g., calcitoninetidronate disodium, levothyroxine Na, liothyronine Na, liotrix,thyroglobulin, teriparatide acetate; antithyroid drugs; estrogenichormones; progestins and antagonists; hormonal contraceptives;testicular hormones; gastrointestinal hormones, e.g., cholecystokinin,enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growthfactor-urogastrone, gastric inhibitory polypeptide, gastrin-releasingpeptide, gastrins, pentagastrin, tetragastrin, motilin, peptide YY,secretin, vasoactive intestinal peptide, or sincalide.

Still other examples of biologically active cargo that can be deliveredaccording to the present disclosure include, but are not limited to,enzymes such as hyaluronidase, streptokinase, tissue plasminogenactivator, urokinase, PGE-adenosine deaminase; intravenous anestheticssuch as droperidol, etomidate, fetanyl citrate/droperidol, hexobarbital,ketamine HCl, methohexital Na, thiamylal Na, thiopental Na;antiepileptics, e.g., carbamazepine, clonazepam, divalproex Na,ethosuximide, mephenyloin, paramethadione, phenyloin, primidone. Invarious embodiments, the biologically active cargo is an enzyme selectedfrom hyaluronidase, streptokinase, tissue plasminogen activator,urokinase, PGE-adenosine deaminase.

Yet other examples of biologically active cargo that can be deliveredaccording to the present disclosure include, but are not limited to,chemotherapeutics, such as chemotherapy or anti-tumor agents which areeffective against various types of human cancers, including leukemia,lymphomas, carcinomas, sarcomas, myelomas etc., such as, for example,doxorubicin, mitomycin, cisplatin, daunorubicin, bleomycin, actinomycinD, and neocarzinostatin.

Modulators of Inflammation (Interleukin-10 and Related Cytokines)

Interleukin-10 (IL-10) is an important immunoregulatory cytokineproduced by many cell populations and whose main biological functionseems to be the limitation and termination of inflammatory responses andthe regulation of differentiation and proliferation of several immunecells such as T cells, B cells, natural killer cells, antigen-presentingcells, mast cells, and granulocytes. More recent data suggests thatIL-10 also mediates immunostimulatory properties that help to eliminateinfectious and noninfectious particles with limited inflammation;Asadullah et al., Pharmacol Rev, 55:241-269, 2003. Moreover, numerousinvestigations suggest a major impact of IL-10 in inflammatory,malignant, and autoimmune diseases, and IL-10 overexpression was foundin certain tumors such as melanoma, basal cell and squamous cellcarcinoma and several lymphomas; Id. Five new human moleculesstructurally related to IL-10 have been discovered, IL-19 (Gallagher etal., Genes Immun., 1:442-450, 2000); IL-20 (Blumberg et al., Cell,104:9-19, 2001), IL-22 (Dumoutier et al., Genes Immun., 1:488-494,2000), IL-24 (Jiang et al., Oncogene, 11:2477-2486, 1995) and IL-26(Knappe et al., J. Virol., 74:3881-3887, 2000) and data suggests thatimmune cells are a major source of the new IL-10 family members; Wolk etal., J. Immunol., 168:5397-5402, 2002.

While there were some promising results from IL-10 delivery on thecourse of several inflammatory diseases in experimental models, severalclinical studies evaluating IL-10 as a therapeutic agent for thetreatment of inflammatory and/or immune disorders remain somewhatdisappointing, with much of the data conflicting; Asadullah et al.,Pharmacol Rev, 55:241-269, 2003. Overall, the data suggests that IL-10is safe and generally well tolerated, however, the ultimate local IL-10concentration in the intestine after systemic administration withstandard doses is too low, resulting in only marginal efficacy. Id.Unfortunately, the ability to sufficiently increase the doses is limiteddue to side effects (e.g., anemia, headache), and there are concernshigher doses of systemically administered IL-10 may be detrimentalrather than helpful in certain indications, e.g., Crohn's; Herfarth etal, Gut, 50(2): 146-147, 2002.

In various embodiments, the biologically active cargo is a polypeptidethat has been determined to be a modulator of inflammation in the GItract selected from, e.g., interleukin-10, interleukin-19,interleukin-20, interleukin-22, interleukin-24, or interleukin-26.

Interleukin-10 (IL-10) was first identified as a product of the type 2helper T cell and later shown to be produced by other cell typesincluding B cells and macrophages (Moore et al., Annu Rev Immunol,19:683-765, 2001). It also inhibits the synthesis of several cytokinesproduced from type 1 helper T cells, such as γ-interferon, IL-2, andtumor necrosis factor-α (TNF-α) (Fiorentino et al., J Immunol,146:3444-3451, 1991). The ability of IL-10 to inhibit cell-mediatedimmune response modulators and suppress antigen-presentingcell-dependent T cell responses demonstrates IL-10 has immunosuppressiveproperties. This cytokine also inhibits monocyte/macrophage productionof other cytokines such as IL-1, IL-6, IL-8, granulocyte-macrophagecolony-stimulating factor (GM-CSF), granulocyte colony-stimulatingfactor (G-CSF), and TNF-α.

The IL-10 protein forms a functional dimer that becomes biologicallyinactive upon disruption of the non-covalent interactions connecting itstwo monomer subunits. The N-terminus does not appear to be directlyinvolved with IL-10 receptor activation. Thus, in one aspect of thedisclosure, a fusion molecule is constructed via conjugation through theN-terminus of the IL-10 protein to the C-terminus of a modified Cholixtoxin using a cleavable linker. Such a construction may result in asolution dimer as a result of IL-10 interactions.

In various embodiments, the biologically active cargo is humaninterleukin-10 having the amino acid sequence set forth in SEQ ID NO:82:

(SEQ ID NO: 82) MHSSALLCCLVLLTGVRASPGQGTOSENSCTHFPGNLPNMLRDLRDAFSRVKIFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN or a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 82.

IL-19 a cytokine that belongs to the IL-10 cytokine subfamily. Thiscytokine is found to be preferentially expressed in monocytes. It canbind the IL-20 receptor complex and lead to the activation of the signaltransducer and activator of transcription 3 (STAT3) (Yamamoto-Furusho JK, et al. Hum Immunol, 72(11):1029-32, 2011). In various embodiments,the biologically active cargo is human interleukin-19 having the aminoacid sequence set forth in SEQ ID NO: 83:

(SEQ ID NO: 83) MKLQCVSLWLLGTILILCSVDNHGLRRCLISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVI HDNYDQLEVHAAAIKSLGELDVFLAWINKNHEVMSSAor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 83.

IL-20 is a cytokine structurally related to interleukin 10 (IL-10). Thiscytokine has been shown to transduce its signal through signaltransducer and activator of transcription 3 (STAT3) in keratinocytes. Aspecific receptor for this cytokine is found to be expressed in skin andupregulated dramatically in psoriatic skin, suggesting a role for thisprotein in epidermal function and psoriasis (Yamamoto-Furusho J K, etal. Immunol Lett, 149(1-2):50-3 2013). In various embodiments, thebiologically active cargo is human interleukin-20 having the amino acidsequence set forth in SEQ ID NO: 84:

(SEQ ID NO: 84) MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSCVIATNLQEIRNGFSEIRGSVQAKDGNIDIRILRRTESLQDTKPANRCCLLRHLLRLYLDRVFKNYQTPDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMK KYSQILSHFEKLEPQAAVVKALGELDILLQWMEETEor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 84.

IL-22 is a cytokine structurally related to interleukin 10 (IL-10).IL-22 secreting CD4(+) T (Th22) cells and IL-22 are involved in thepathogenesis of autoimmune disease, and may play an important role inthe pathogenesis of NMO and MS (Xu et al., J Neuroimmunol., August 15;261(1-2):87-91, 2013). In various embodiments, the biologically activecargo is human interleukin-22 having the amino acid sequence set forthin SEQ ID NO: 85:

(SEQ ID NO: 85) MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACIor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 85.

IL-24 is a cytokine structurally related to interleukin 10 (IL-10) whichcan induce apoptosis selectively in various cancer cells. Overexpressionof this gene leads to elevated expression of several GADD family genes,which correlates with the induction of apoptosis. The phosphorylation ofmitogen-activated protein kinase 14 (MAPK7/P38), and heat shock 27 kDaprotein 1 (HSPB2/HSP27) are found to be induced by this gene in melanomacells, but not in normal immortal melanocytes (Lin B W, et al., J KoreanMed Sci, 28(6):833-9, 2013). In various embodiments, the biologicallyactive cargo is human interleukin-24 having the amino acid sequence setforth in SEQ ID NO: 86:

(SEQ ID NO: 86) MNFQQRLQSLWTLASRPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTW MQKFYKLor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 86.

IL-26 was identified by its overexpression specifically in herpesvirussaimiri-transformed T cells. The encoded protein is a member of theIL-10 family of cytokines. It is a secreted protein and may function asa homodimer. This protein is thought to contribute to the transformedphenotype of T cells after infection by herpesvirus saimiri (CorvaisierM, et al. PLoS Biol, 10(9):e1001395, 2012). In various embodiments, thebiologically active cargo is human interleukin-26 having the amino acidsequence set forth in SEQ ID NO: 87:

(SEQ ID NO: 87) MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIY KAISELDILLSWIKKLLESSQor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 87.

Importantly, the non-naturally occurring fusion molecules which lack acleavable linker can be advantageous in that the anchoring effect of themodified Cholix toxin by its receptor(s) at the surface of, e.g., immunecells that also express the receptor for the IL-10 (but in considerablylower quantity) can allow for greater exposure of the IL-10 at thesurface of the targeted cells, and provide a synergistic effect via thebinding of the Cholix to its receptor and the binding of IL-10 to theIL-10R.

Tumor Necrosis Factor Super Family

Tumor necrosis factor is a rapidly growing superfamily of cytokines(hereinafter “TNFSF”) that interact with a corresponding superfamily ofreceptors (hereinafter “TNFSFR”). Since the discovery of tumor necrosisfactor-alpha (“TNF-α”) about 25 years ago, the TNFSF has grown to alarge family of related proteins consisting of over 20 members thatsignal through over 30 receptors (see, e.g., “Therapeutic Targets of theTNF Superfamily”, edited by Iqbal S. Grewal, Landes Bioscience/SpringerScience+Business Media, LLC dual imprint/Springer series: Advances inExperimental Medicine and Biology, 2009). Members of TNFSF have widetissue distribution and TNFSF ligand-receptor interactions are involvedin numerous biological processes, ranging from hematopoiesis topleiotropic cellular responses, including activation, proliferation,differentiation, and apoptosis. TNFSF ligand-receptor interactions havealso been implicated in tumorigenesis, transplant rejection, septicshock, viral replication, bone resorption and autoimmunity. Theparticular response depends upon the receptor that is signaling, thecell type, and the concurrent signals received by the cell.

Because a number of TNFSF members are expressed on tumor cells, antibodybased therapies are being developed to target these molecules and someare currently undergoing clinical trials (e.g., TNF-α for human use inthe treatment of sarcomas and melanomas (Eggermont et al., Lancet Oncol,4:429-437, 2003; Lans et al., Clin Cancer Res, 7:784-790, 2001). Inaddition, many of these molecules are also being exploited as targetsfor antibody-drug conjugates (e.g., CD30 and CD70), or exploited forradioimmunotherapy (e.g., the BLyS receptors TACI and BR3) (Buchsbaum etal., J Nucl Med, 44:434-436, 2003).

Similarly, because a number of TNFSF members have been implicated inboth innate and adaptive immune responses such as defense againstpathogens, inflammatory response and autoimmunity, approaches to targetmany of TNFSF receptors and ligands for treatment of autoimmunity andother inflammatory diseases are being exploited. Indeed, a number ofbiologic TNF blocking therapies (hereinafter “TNF inhibitors”) includinghumanized/human monoclonal antibodies (e.g., infliximab (REMICADE®) oradalimumab (HUMIRA®)) or recombinant fusion proteins of IgG and solubleTNFSF receptors (e.g., etanercept (ENBREL®)) have been developed and arenow being used in humans to inhibit the inflammation associated withCrohn's disease and rheumatoid arthritis (Mitoma et al., ArthritisRheum, 58:1248-1257, 2008; Shealy et al., Handb Exp Pharmacol,181:101-129, 2008). Thus, the potential to deliver such agents locallyincluding, but not limited to, intestinal and pulmonary mucosa, wouldprovide added benefits for efficacy and safety.

Although these various TNF inhibitors have been approved for humantherapies and are being successfully used in human patients, thereremains a number of toxicities associated with these TNF inhibitors,e.g., hepatotoxicity, thromboembolic complications, and increased riskof development of tuberculosis and lymphoma (Gardam et al., LancetInfect Dis, 3:148-155, 2003). Moreover, while effective in haltingprogression of disease, these agents are very expensive, generallyadministered intravenously or subcutaneously, and do not cure thediseases. The continued examination of signal transduction of TNFSFmembers is needed to develop approaches for tissue specificinterventions, which could allow targeted therapies to have fewer sideeffects.

In various embodiments, the biologically active cargo is a TNF inhibitorthat is an isolated antibody or an antibody fragment. Isolatedantibodies and antibody fragments useful in the constructs and methodsof the present invention include, without limitation, monoclonal Abs(mAbs), polyclonal Abs, Ab fragments (e.g., Fab, Fab′, F(ab′)2, Fv, Fc,etc.), chimeric Abs, mini-Abs or domain Abs (dAbs), dual specific Abs,bispecific Abs, heteroconjugate Abs, single chain Abs (SCA), singlechain variable region fragments (ScFv), fusion proteins comprising an Abportion or multiple Ab portions, humanized Abs, fully human Abs, and anyother modified configuration of the immunoglobulin (Ig) molecule thatcomprises an antigen recognition site of the required specificity.

Anti-TNF-α Antibodies. The FDA approved anti-TNF-α antibody, Adalimumab(Abbvie HUMIRA®; DrugBank DB 00051) has been used to treat humans. Invarious embodiments of the present invention, the biologically activecargo is a human antibody or antigen-binding fragment comprising theheavy chain variable region sequence set forth in SEQ ID NO: 88:

(SEQ ID NO: 88) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVERGFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCand the light chain variable region sequence set forth in SEQ ID NO:89:

(SEQ ID NO: 89) DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECor an antigen-binding or an immunologically functional immunoglobulinfragment thereof.

In various embodiments, the invention provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable region, and wherein the heavy chain variable regioncomprises a sequence that has at least about 75%, at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO:88; and wherein the light chain comprises a lightchain variable region, and wherein the light chain variable regioncomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in any of SEQID NO:89; wherein the antibody binds specifically to human TNF-α.

The FDA approved anti-TNF-a antibody, Infliximab (Centocor REMICADE®;Drug Bank DB 00065) has been used to treat humans. In variousembodiments of the present invention, the biologically active cargo is ahuman antibody or antigen-binding fragment comprising the heavy chainvariable region sequence set forth in SEQ ID NO: 90:

(SEQ ID NO: 90) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAIISFDGSNKSSADSVKGRFTUSRRNSKNALFLQMNSLRAEDTAVFYCARDRGVSAGGNYYYYGMDVWGQGTTVTVSSand the light chain variable region sequence set forth in SEQ ID NO:91:

(SEQ ID NO: 91) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTRFTLTISSLEPEDFAVYYC QQRSNWPPFTFGPGTKVDILor an antigen-binding or an immunologically functional immunoglobulinfragment thereof.

In various embodiments, the invention provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a heavychain variable region, and wherein the heavy chain variable regioncomprises a sequence that has at least about 75%, at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO:90; and wherein the light chain comprises a lightchain variable region, and wherein the light chain variable regioncomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in any of SEQID NO:91; wherein the antibody binds specifically to human TNF-α.

Antibodies to several other TNFSF ligands or TNFSFRs have been describedin the literature, and evaluated as therapeutic candidates in thetreatment or prevention of a variety of inflammatory diseases,autoimmune diseases and cancer. Nucleotide and amino acid sequences ofantibodies to the designated TNFSF polypeptides or TNFSFRs are readilyavailable from publicly available databases. A comprehensive review ofsuch antibodies as well as additional TNF inhibitors is provided in“Therapeutic Targets of the TNF Superfamily”, edited by Iqbal S. Grewal,Landes Bioscience/Springer Science+Business Media, LLC dualimprint/Springer series: Advances in Experimental Medicine and Biology,2009, which is hereby incorporated by reference in its entirety for thepurpose of teaching such TNF inhibitors.

In various embodiments, the biologically active cargo is a TNFSFinhibitor that comprises a soluble receptor or soluble co-ligand. Theterms “soluble receptor”, “soluble cytokine receptor” (SCR) and“immunoadhesin” are used interchangeably to refer to soluble chimericmolecules comprising the extracellular domain of a receptor, e.g., areceptor of a TNFSF member and an Ig sequence, which retains the bindingspecificity of the receptor and is capable of binding to the TNFSFmember. In various embodiments, a TNFSFSCR comprises a fusion of aTNFSFR amino acid sequence (or a portion thereof) from a TNFSF memberextracellular domain capable of binding the TNFSF member (in someembodiments, an amino acid sequence that substantially retains thebinding specificity of the TNFSFR) and an Ig sequence. Two distincttypes of TNFSFR are known to exist: Type I TNFSFR (TNFSFRI) and Type IITNFSFR (TNFSFRII). In various embodiments, the TNFSF receptor is a humanTNFSF receptor sequence, and the fusion is with an Ig constant domainsequence. In other embodiments, the Ig constant domain sequence is an Igheavy chain constant domain sequence. In other embodiments, theassociation of two TNF receptor-Ig heavy chain fusions (e.g., viacovalent linkage by disulfide bond(s)) results in a homodimeric Ig-likestructure.

An example of a commercially available soluble receptor useful in thepresent invention is ENBREL® (etanercept). ENBREL® consists ofrecombinant human TNFR-p75-Fc dimeric fusion protein consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgG1. The Fc component of etanercept contains the CH2 domain, the CH3domain and hinge region, but not the CH1 domain of IgG1. Etanercept isproduced by recombinant DNA technology in a Chinese hamster ovary (CHO)mammalian cell expression system. It consists of 934 amino acids. Theproduct is made by encoding the DNA of the soluble portion of humanTNFR-p75 with the Fc portion of IgG. In various embodiments of thepresent invention, the biologically active cargo is a TNF inhibitor thatis dimeric fusion protein comprising the sequence set forth in SEQ IDNO: 92:

(SEQ ID NO: 92) LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of at least about 75%, atleast about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the sequenceof SEQ ID NO: 92.

An illustrative, but not limiting list of suitable TNFSF ligands andTNFSFRs from which a TNF inhibitor will be derived and used as abiologically active cargo in the constructs and methods of the presentinvention is provided in Table 2.

TABLE 2 RefSeq (protein) TNFSF Ligands Tumor necrosis factor-α (“TNF-α”)NP_000585.2 lymphotoxin-α (“LT-α”) NP_000586.2 lymphotoxin-β (“LT-β”)NP_002332.1 CD30 ligand NP_001235.1 CD40 ligand NP_000065.1 CD70 ligandNP_001243.1 OX40 ligand NP_001284491.1 41BB ligand NP_001552.2 Apo1ligand (or FasL or CD95L) NP_000630.1 Apo2 ligand (or TRAIL, AIM-1 orAGP-1) NP_001177871.1 Apo3 ligand (or TWEAK) NP_003800.1 APRILNP_001185551.1 LIGHT NP_003798.2 OPG ligand (or RANK ligand) NP_003692.1BlyS (or THANK) NP_001139117.1 BCMA NP_001183.2 TACI NP_036584.1 TNFSFRsTNFR1 NP_001056.1 TNFR2 NP_001057.1 lymphotoxin-βR NP_001257916.1 CD40NP_001241.1 CD95 (or FAS or APO-1) NP_000034.1 OPG NP_002537.3 RANKNP_001257878.1 CD30 NP_001234.3 CD27 NP_001233.1 OX40 (or CD134)NP_003318.1 41BB NP_001552.2 NGFR NP_002498.1 BCMA NP_001183.2 TAC1NP_036584.1 EDA2R NP_001186616.1 TROY NP_001191387.1 DR6 NP_055267.1 DR5(or TRAILR2) NP_003833.4 DR4 NP_003835.3 DR3 NP_001034753.1 HVEMNP_001284534.1 LTβR NP_001257916.1 GITR NP_004186.1 DcR3 NP_003814 Fn14(or TWEAKR) NP_057723.1 BAFF NP_443177.1

Glucose-Lowering Agents

In various embodiments, the biologically active cargo is aglucose-lowering agent. In various embodiments, the glucose-loweringagent is a peptide that comprises about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 25, about 30,about 35, about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95, about 100,about 150, about 200, about 250, about 300, about 400, about 500, about600, about 700, about 800, about 900 or about 1000 amino acids.

An illustrative, but not limiting, list of suitable glucosemetabolism-related proteins to be used as the glucose-lowering agent inthe fusion molecules of the present disclosure, or from which theglucose-lowering agents contemplated for use as a glucose-lowering agentcould be derived, is provided in Table 3.

TABLE 3 Glucose metabolism-related proteins RefSeq (NCBI/Uniprot)Glucagon proprotein NP_002045.1 Glucagon peptide NP_002045.1 (aa 53-81)Glucagon-like peptide 1 NP_002045.1 (aa 98-128) Glucagon-like peptide 2NP_002045.1 (aa 146-178) Glicentin P01275 (aa 21-89) Glicentin-relatedpolypeptide P01275 (aa 21-50) Gastric inhibitory polypeptide NP_004114.1preprotein Gastric inhibitory polypeptide NP_004114.1 (aa 52-93)Dipeptidyl peptidase 4 P27487 Glucose transporter member 4 NP_001033.1Preproglucagon AAA52567.1 Insulin receptor substrate 1 NP_005535.1Insulin P01308 Apolipoprotein A-II P02652 Solute carrier family 2,faciliated P11166 glucose transporter member 1 Glycogen synthase 1P13807 Glycogen synthase 2 P54840 Tyrosin-protein phosphatase non-P18031 receptor type 1 RAC-alpha serinel threonine- P31749 proteinkinase Peroxisome proliferator-activated P37231 receptor gammaHexokinase 3 P52790 Phosphatidylinositol-3,4,5- P60484 triphosphate3-phosphatase and dual-specificty protein Pyruvate dehydrogenase kinase1 Q15118 Calcium-binding and coiled-coil Q9P1Z2 domain-containingprotein 1 Max-like protein X Q9UH92 Fructose-bisphosphate aldolase AP04075 Glucagon-like peptide 1 receptor P43220 Glucagon-like peptide 2receptor O95838 Gastric inhibitory polypeptide P48546 receptorInsulin-like growth factor 1 P08069.1 receptor Insulin-like growthfactor 2 P11717.3 receptor Insulin Receptor P06213 GLP-1agonist-Exenatide DB01276 GLP-1 agonist-Liraglutide DB06655

Glucagon-like peptide-1 (GLP-1), a member of the pro-glucagon incretinfamily synthesized in intestinal L-cells by tissue-specificpost-translational processing of the glucagon precursor preproglucagon,is a potent glucose-lowering agent implicated in the control of appetiteand satiety. GLP-1 acts through GLP-1 receptor (GLP-1 R), which iswidely distributed in tissues, including brain, pancreas, intestine,lung, stomach, and kidney. The effects of GLP-1 appear to be bothinsulinotropic and insulinomimetic, depending on the ambient glucoseconcentration. Due to their ability to increase insulin secretion fromthe pancreas, increase insulin-sensitivity in both alpha cells and betacells, and decrease glucagon secretion from the pancreas, GLP-1 and itsanalogs have attracted considerable attention as a therapeutic strategyfor diabetes.

Several clinical trials have studied the addition of GLP-1 agonists inconjunction with ongoing insulin therapy and several GLP-1 agonists havebeen approved for treatment of T2 D, including, e.g., exenatide(tradename Byetta®, Amylin/Astrazeneca); liraglutide (tradenameVictoza®, Novo Nordisk A/S); lixisenatide (tradename Lyxumia®, Sanofi);albiglutide (tradename Tanzeum®, GlaxoSmithKline); dulaglutide(tradename Trulicity®, Eli Lilly). While proven efficacious, the majordrawback associated with the clinical use of GLP-1 agonists is the shortbiological half-life, necessitating continuous administrationintravenously or by frequent subcutaneous injections, and all GLP-1drugs approved to date are subcutaneous administered on a twice daily oronce weekly basis. Moreover, there are safety concerns associated withthe use of these GLP-1 agonists, namely, pancreatitis and pancreaticneoplasia, hypoglycemia, and renal impairment. Other reported sideeffects include gastrointestinal disorders, such as dyspepsia, decreasedappetite, nausea, vomiting, abdominal pain, diarrhea, dizziness,headache, and feeling jittery. As such, there continues to be extensiveresearch directed to preparing analogs of the natural GLP-1 that arelonger lasting, as well as development of sustained release and otherrelated technologies in order to lower the frequency of injections forthe T2D patients.

In various embodiments, the biologically active cargo is GLP-1 agonisthaving the amino acid sequence set forth in SEQ ID NO: 93:

(SEQ ID NO: 93) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 93.

In various embodiments, the biologically active cargo is GLP-1 agonisthaving the amino acid sequence set forth in SEQ ID NO: 94:

(SEQ ID NO: 94) HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRGor a fragment or variant thereof.

In various embodiments, the biologically active cargo contains an aminoacid sequence that shares an observed homology of, e.g., at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% with thesequence of SEQ ID NO: 94.

Human Growth Hormone

Growth Hormone (GH) (also known as somatropin or somatotropin) is themaster hormone in the human body, and is synthesized and secreted by theendocrinal system. This hormone controls essential functions like:growth and replication of cells in various organs of the body. Some ofthe essential functions of GH include: controlling muscle growth,improving bone mineralization and strength, reducing fat deposition, andsustaining good energy levels. The production and secretion of thegrowth hormone is controlled by Growth Hormone Releasing Hormone (GHRH),which is secreted by the hypothalamus. The GHRH stimulates the pituitarygland to produce GH, which is directly released into the blood stream.The GH in turn stimulates the liver to produce Insulin-like GrowthFactor (IGF-1) which stimulates the proliferation of chondrocytes(cartilaginous cells), promotes differentiation of myoblasts andenhances protein synthesis, which in turn, helps in the growth of othermuscles and tissue cells.

In the US, synthetically produced human growth hormone (HGH) has beenused in the pediatric population to treat short stature due to growthhormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome,Prader-Willi syndrome, short stature homeobox-containing gene (SHOX)deficiency, chronic renal insufficiency, idiopathic short stature andchildren small for gestational age. In adults, HGH has been used totreat short bowel syndrome, a condition in which nutrients are notproperly absorbed due to sever intestinal disease or the surgicalremoval of a large portion of the small intestine, GH deficiency due torare pituitary tumors or their treatment, and muscle-wasting diseaseassociated with HIV/AIDS.

Growth hormone deficiency (GHD) is a rare disorder that includes a groupof different pathologies characterized by the inadequate secretion ofgrowth hormone (GH) from the anterior pituitary gland, a small glandlocated at the base of the brain that is responsible for the productionof several hormones. GHD may occur by itself or in combination withother pituitary hormone deficiencies. GHD may be present from birth(congenital) or acquired as a result of trauma, infiltrations, tumor orradiation therapy. There is a third category that has no known cause(idiopathic). Childhood-onset GHD may be all three: congenital,acquired, or idiopathic. It results in growth retardation, shortstature, and maturation delays reflected by the delay of lengthening ofthe bones of the extremities that is inappropriate to the chronologicalage of the child. Adult-onset GHD is most often acquired from apituitary tumor or trauma to the brain but may also be idiopathic. It ischaracterized by a number of variable symptoms including reduced energylevels, altered body composition, osteoporosis (reduced bone mineraldensity), reduced muscle strength, lipid abnormalities such as increasedLDL or cholesterol levels, insulin resistance, and impaired cardiacfunction. Adult GHD has been estimated to affect 1 in 100,000 peopleannually, while its incidence rate is approximately 2 cases per 100,000population when childhood-onset GHD patients are considered. About15-20% of the cases represent the transition of childhood GHD intoadulthood (Stochholm K et al., Eur J Endocrinol., 155:61-71, 2006).

Turner (or Ullrich-Turner) syndrome (TS) is a chromosomal abnormalitycharacterized by the absence of the entire chromosome X or a deletionwithin that chromosome and that affects development in females. The mostcommon feature of Turner syndrome is short stature, which becomesevident by about age 5. This condition occurs in about 1 in 2,500newborn girls worldwide, but it is much more common among pregnanciesthat do not survive to term (miscarriages and stillbirths). As achromosomal condition, there is no cure for Turner syndrome.

Recombinant DNA-derived human growth hormone is the only drug approvedspecifically for treatment of GHD and TS. As of 2005, variousrecombinant human growth hormones (also referred to as somatropin [rDNAorigin] for injection) available in the United States (and theirmanufacturers) included NUTROPIN® (Genentech), HUMATROPE® (Lilly),GENOTROPIN® (Pfizer), NORDITROPIN® (Novo), and SAIZEN® (Merck Serono).In 2006, the U.S. Food and Drug Administration (FDA) approved a versionof rHGH called OMNITROPE® (Sandoz). A sustained-release form of humangrowth hormone, NUTROPIN DEPOT® (Genentech/Alkermes) was approved by theFDA in 1999, allowing for fewer injections (every 2 or 4 weeks insteadof daily); however, the product was discontinued by Genentech/Alkermesin 2004 for financial reasons. Additional approved recombinant HGHproducts include SEROSTIM® (EMD Serono), TEV-TROPIN® (Teva) andZORBITIVE® (Merck Serono) for short bowel syndrome.

While proven to be the most effective, spontaneous and trusted treatmentoption for the management of growth disorders such as GHD, theseinjectable rHGH's have some significant limitations including, e.g, 1)complications associated with prolonged use and high dosages which aresevere and irreversible, and include, e.g, the probability of developingdiabetes, cardiovascular disorders and colon cancer. Other common sideeffects include: joint pain, generalized edema, severe headache,hypoglycemia, wrist pain (carpel tunnel syndrome), increased level ofLDL in the blood increasing the possibility of developingatherosclerosis, etc.; 2) HGH injections are not available over thecounter, nevertheless, due to rigid FDA norms, black-marketing isrampant. The procurement of the HGH injections without medicalprescription is considered illegal and is punishable by law, withimprisonment and fine; and 3) the cost of the treatment is exorbitant.Depending upon the pharmaceutical company the cost of HGH injections fora month of treatment, typically range from between $800 to $3000.Finally, conventional methods using rHGH typically involve multi-doseregimens in which the HGH is administered via subcutaneous injection.The inconvenience, pain and social stigma associated with such methodscan be considerable. Management of the pediatric population to treatshort stature due to growth hormone deficiency (GHD), Turner syndrome(TS) and related disorders, with these highly invasive and repetitivetherapies can be especially difficult.

Full length human HGH consists of 191 amino acids. HGH produced usingmolecular biological techniques may have an amino acid sequenceidentical to naturally occurring HGH. Alternatively, the HGH used may bean HGH analog comprising one or more variations in amino acid sequencewith respect to the native hormone. These amino acid variations mayprovide enhanced biological activity or some other biological orlogistical advantages. In various embodiments, the recombinant HGHcomprises the amino acid sequence set forth in Genbank Accession No.P01241. The HGH amino acid sequence (without the 26 aa signal sequenceof P01241) is set forth in SEQ ID NO: 95:

(SEQ ID NO: 95) FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

HGH of the present disclosure refers to HGH from any source which hasthe sequence of SEQ ID NO: 95, including isolated, purified and/orrecombinant HGH produced from any source or chemically synthesizes, forexample using solid phase synthesis. Also included herein are conservedamino acid substitutions of native HGH. For example, conservative aminoacid changes may be made, which although they alter the primary sequenceof the protein or peptide, do not normally alter its function.Conservative amino acid substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. In variousembodiments, the HGH has an amino acid sequence that shares an observedhomology of, e.g., at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or at least about 99% with the sequence of SEQ ID NO: 95.

In various embodiments, the HGH contemplated for use in the fusionmolecules of the present disclosure include human growth hormonevariants and mutants which have been extensively described in the art(see, e.g. U.S. Pat. No. 8,637,646 (Wells et al) and references citedtherein, and US 20110130331 (Guyon et al), each incorporated byreference in its entirety herein for the specific purpose of providingsuch growth hormone variants and mutants).

In various embodiments, the HGH contemplated for use in the fusionmolecules of the present disclosure include, e.g., NUTROPIN®(Genentech), HUMATROPE® (Lilly), GENOTROPIN® (Pfizer), NORDITROPIN®(Novo), SAIZEN® (Merck Serono), OMNITROPE® (Sandoz), SEROSTIM® (EMDSerono), TEV-TROPIN® (Teva) and ZORBITIVE® (Merck Serono).

An illustrative, but not limiting, list of suitable growth hormoneproteins to be used as the growth hormone in the fusion molecules of thepresent disclosure, or from which the growth hormones contemplated foruse as a growth hormone could be derived, is provided in Table 4.

TABLE 4 RefSeq (NCBI/Uniprot) Growth Hormone Related ProteinsSomatotropin P01241 Synthetic Human Growth Hormone AAA72260.1 SyntheticHuman Growth Hormone CAA01435 Partial Synthetic Human Growth HormoneCAA00380 Partial Human Growth Hormone 2 P01242 Somatoliberin P01286.1Appetite-regulating Hormone Q9UBU3 Leptin P41159 Growth Hormone ReceptorProteins Growth Hormone Receptor P10912 Growth Hormone-Releasing HormoneQ02643 Receptor Growth Hormone Secretagogue Q92847 Receptor GrowthHormone-Releasing Hormone P78470 Receptor form a Growth Hormone ReceptorE9PCN7

Insertion Site for Attachment of the Biologically Active Cargo

The biologically active cargo of the fusion molecule can be attached tothe remainder of the fusion molecule by any method known by one of skillin the art without limitation. The biologically active cargo can beintroduced into any portion of the fusion molecule that does not disruptthe cell-binding or transcytosis activity of the modified Cholix toxin.In various embodiments, the biologically active cargo is directlycoupled to the N-terminus or C-terminus of the modified Cholix toxin. Invarious embodiments, the biologically active cargo can be connected witha side chain of an amino acid of the modified Cholix toxin. In variousembodiments, the biologically active cargo is coupled to the modifiedCholix with a non-cleavable peptide linker. In various embodiments, thebiologically active cargo is coupled to the modified Cholix toxin with acleavable linker such that cleavage at the cleavable linker(s) separatesthe biologically active cargo from the remainder of the fusion molecule.In various embodiments, the biologically active cargo is a polypeptidethat may also comprise a short leader peptide that remains attached tothe polypeptide following cleavage of the cleavable linker. For example,the biological active cargo can comprise a short leader peptide ofgreater than 1 amino acid, greater than 5 amino acids, greater than 10amino acids, greater than 15 amino acids, greater than 20 amino acids,greater than 25 amino acids, greater than 30 amino acids, greater than50 amino acids, or greater than 100 amino acids. In some cases,biological active cargo can comprise a short leader peptide of less than100 amino acids, less than 50 amino acids, less than 30 amino acids,less than 25 amino acids, less than 20 amino acids, less than 15 aminoacids, less than 10 amino acids, or less than 5 amino acids. In somecases, biological active cargo can comprise a short leader peptide ofbetween 1-100 amino acids, between 5-10 amino acids, between 10 to 50amino acids, or between 20 to 80 amino acids. In native Cholix toxin,the domain Ib loop spans amino acids 387 to 425, and is structurallycharacterized by a disulfide bond between two cysteines at positions 395and 402. This domain Ib portion of Cholix toxin is not essential for anyknown activity of Cholix toxin, including cell binding, translocation,ER retention or ADP ribosylation activity. Accordingly, domain Ib can bedeleted entirely, or modified to contain a biologically active cargo.Thus, in various embodiments, the biologically active cargo can beinserted into Cholix toxin domain Ib. If desirable, the biologicallyactive cargo can be inserted into Cholix toxin domain Ib between thecysteines at positions 395 and 402 that are not crosslinked. This can beaccomplished by reducing the disulfide linkage between the cysteines, bydeleting one or both of the cysteines entirely from the Ib domain, bymutating one or both of the cysteines to other residues, for example,serine, or by other similar techniques. Alternatively, the biologicallyactive cargo can be inserted into the domain Ib loop between thecysteines at positions 395 and 402. In such embodiments, the disulfidelinkage between the cysteines can be used to constrain the biologicallyactive cargo domain.

In embodiments where the biologically active cargo is expressed togetherwith another portion of the fusion molecule as a fusion protein, thebiologically active cargo can be can be inserted into the fusionmolecule by any method known to one of skill in the art withoutlimitation. For example, amino acids corresponding to the biologicallyactive cargo can be directly inserted into the fusion molecule, with orwithout deletion of native amino acid sequences. In various embodiments,all or part of the Ib domain of Cholix toxin can be deleted and replacedwith the biologically active cargo. In various embodiments, the cysteineresidues of the Ib loop are deleted so that the biologically activecargo remains unconstrained. In other embodiments, the cysteine residuesof the Ib loop are linked with a disulfide bond and constrain thebiologically active cargo.

In embodiments where the biologically active cargo is not expressedtogether with the remainder of the fusion molecule as a fusion protein,the biologically active cargo can be connected with the remainder of thefusion molecule by any suitable method known by one of skill in the art,without limitation. More specifically, the exemplary methods describedabove for connecting a receptor binding domain to the remainder of themolecule are equally applicable for connecting the biologically activecargo to the remainder of the molecule.

Production of Fusion Proteins

In various embodiments, the non-naturally occurring fusion molecule issynthesized using recombinant DNA methodology. Generally this involvescreating a DNA sequence that encodes the fusion molecule, placing theDNA in an expression cassette under the control of a particularpromoter, expressing the molecule in a host, isolating the expressedmolecule and, if required, renaturing the molecule.

DNA encoding the fusion molecules (e.g. Cholix⁴¹⁵-IL-10) describedherein can be prepared by any suitable method, including, for example,cloning and restriction of appropriate sequences or direct chemicalsynthesis by methods such as the phosphotriester method of Narang et al.(1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown etal. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite methodof Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solidsupport method of U.S. Pat. No. 4,458,066, and the like.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In various embodiments, DNA encoding fusion molecules of the presentdisclosure can be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the gene for theIL-10 is PCR amplified, using a sense primer containing the restrictionsite for, e.g., NdeI and an antisense primer containing the restrictionsite for HindIII. This can produce a nucleic acid encoding the matureIL-10 sequence and having terminal restriction sites. A modified Cholixtoxin having “complementary” restriction sites can similarly be clonedand then ligated to the IL-10 and/or to a linker attached to the IL-10.Ligation of the nucleic acid sequences and insertion into a vectorproduces a vector encoding the IL-10 joined to the modified Cholixtoxin.

Non-Cleavable Linkers

In various embodiments, the modified Cholix toxin and biologicallyactive cargo can be separated by a peptide spacer consisting of one ormore amino acids (e.g., up to 25 amino acids). Generally the spacer willhave no specific biological activity other than to join the proteins orto preserve some minimum distance or other spatial relationship betweenthem. In various embodiments, however, the constituent amino acids ofthe spacer can be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

In various embodiments, the linker is capable of forming covalent bondsto both the Cholix toxin and to the biologically active cargo. Suitablelinkers are well known to those of skill in the art and include, but arenot limited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. In various embodiments, thelinker(s) can be joined to the constituent amino acids of the Cholixtoxin and/or the biologically active cargo through their side groups(e.g., through a disulfide linkage to cysteine). In various embodiments,the linkers are joined to the alpha carbon amino and/or carboxyl groupsof the terminal amino acids of the Cholix toxin and/or the biologicallyactive cargo.

A bifunctional linker having one functional group reactive with a groupon the Cholix toxin and another group reactive on the biologicallyactive cargo, can be used to form the desired conjugate. Alternatively,derivatization can involve chemical treatment of the targeting moiety.Procedures for generation of, for example, free sulfhydryl groups onpolypeptides, such as antibodies or antibody fragments, are known (SeeU.S. Pat. No. 4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075.

In various embodiments, the biologically active cargo to be delivered tothe subject is coupled to the modified Cholix toxin using one or morenon-cleavable peptide linkers comprising, e.g., the amino acid sequenceGGGGS (SEQ ID NO: 96), GGGGSGGGGS (SEQ ID NO: 97), GGGGSGGGGSGGGGS (SEQID NO: 98), or GGGGSGGG (SEQ ID NO: 99), wherein the modified Cholixtoxin targets said biologically active cargo to specific cells,including but not limited to, cells of the immune system such asmacrophages, antigen-presenting cells and dendritic cells.

Cleavable Linkers

In various embodiments, the biologically active cargo to be delivered tothe subject is coupled to the modified Cholix toxin using one or morecleavable linkers. The number of cleavable linkers present in the fusionmolecule depends, at least in part, on the location of the biologicallyactive cargo in relation to the modified Cholix toxin and the nature ofthe biologically active cargo. When the biologically active cargo can beseparated from the remainder of the fusion molecule with cleavage at asingle linker, the fusion molecules can comprise a single cleavablelinker. Further, where the biologically active cargo is, e.g., a dimeror other multimer, each subunit of the biologically active cargo can beseparated from the remainder of the fusion molecule and/or the othersubunits of the biologically active cargo by cleavage at the cleavablelinker.

In various embodiments, the cleavable linkers are cleavable by acleaving enzyme that is present at or near the basolateral membrane ofan epithelial cell. By selecting the cleavable linker to be cleaved bysuch enzymes, the biologically active cargo can be liberated from theremainder of the fusion molecule following transcytosis across themucous membrane and release from the epithelial cell into the cellularmatrix on the basolateral side of the membrane. Further, cleavingenzymes could be used that are present inside the epithelial cell, suchthat the cleavable linker is cleaved prior to release of the fusionmolecule from the basolateral membrane, so long as the cleaving enzymedoes not cleave the fusion molecule before the fusion molecule entersthe trafficking pathway in the polarized epithelial cell that results inrelease of the fusion molecule and biologically active cargo from thebasolateral membrane of the cell.

In various embodiments, the enzyme that is present at a basolateralmembrane of a polarized epithelial cell is selected from, e.g.,Cathepsin GI, Chymotrypsin I, Elastase I, Subtilisin AI, Subtilisin AII,Thrombin I, or Urokinase I. Table 5 presents these enzymes together withan amino acid sequence that is recognized and cleaved by the particularpeptidase.

TABLE 5 Peptidases Present Near Basolateral MucousMembranes or in Latter Aspects of the Transcytosis Pathway Amino AcidPeptidase Sequence Cleaved Cathepsin GI AAPF (SEQ ID NO: 100)Chymotrypsin I GGF (SEQ ID NO: 101) Elastase I AAPV (SEQ ID NO: 102)Subtilisin AI GGL (SEQ ID NO: 103) Subtilisin AII AAL (SEQ ID NO: 104)Thrombin I FVR (SEQ ID NO: 105) Urokinase I VGR (SEQ ID NO: 106) FurinRKPR (SEQ ID NO: 107)

In various embodiments, the cleavable linker exhibits a greaterpropensity for cleavage than the remainder of the delivery construct. Asone skilled in the art is aware, many peptide and polypeptide sequencescan be cleaved by peptidases and proteases. In various embodiments, thecleavable linker is selected to be preferentially cleaved relative toother amino acid sequences present in the delivery construct duringadministration of the delivery construct. In various embodiments, thereceptor binding domain is substantially (e.g., about 99%, about 95%,about 90%, about 85%, about 80, or about 75%) intact following deliveryof the delivery construct to the bloodstream of the subject. In variousembodiments, the translocation domain is substantially (e.g., about 99%,about 95%, about 90%, about 85%, about 80, or about 75%) intactfollowing delivery of the delivery construct to the bloodstream of thesubject. In various embodiments, the macromolecule is substantially(e.g., about 99%, about 95%, about 90%, about 85%, about 80, or about75%) intact following delivery of the delivery construct to thebloodstream of the subject. In various embodiments, the cleavable linkeris substantially (e.g., about 99%, about 95%, about 90%, about 85%,about 80, or about 75%) cleaved following delivery of the deliveryconstruct to the bloodstream of the subject.

In other embodiments, the cleavable linker is cleaved by a cleavingenzyme found in the plasma of the subject. Any cleaving enzyme known byone of skill in the art to be present in the plasma of the subject canbe used to cleave the cleavable linker. Uses of such enzymes to cleavethe cleavable linkers is less preferred than use of cleaving enzymesfound near the basolateral membrane of a polarized epithelial cellbecause it is believed that more efficient cleavage will occur in nearthe basolateral membrane. However, if the skilled artisan determinesthat cleavage mediated by a plasma enzyme is sufficiently efficient toallow cleavage of a sufficient fraction of the delivery constructs toavoid adverse effects, such plasma cleaving enzymes can be used tocleave the delivery constructs. Accordingly, in various embodiments, thecleavable linker can be cleaved with an enzyme that is selected from thegroup consisting of caspase-1, caspase-3, proprotein convertase 1,proprotein convertase 2, proprotein convertase 4, proprotein convertase4 PACE 4, prolyl oligopeptidase, endothelin cleaving enzyme,dipeptidyl-peptidase IV, signal peptidase, neprilysin, renin, andesterase (see, e.g., U.S. Pat. No. 6,673,574, incorporated by referencein its entirety herein). Table 6 presents these enzymes together with anamino acid sequence(s) recognized by the particular peptidase. Thepeptidase cleaves a peptide comprising these sequences at the N-terminalside of the amino acid identified with an asterisk.

TABLE 6 Plasma Peptidases Amino Acid Peptidase Sequence CleavedCaspase-1 Tyr-Val-Ala-Asp-Xaa* (SEQ ID NO: 108) Caspase-3Asp-Xaa-Xaa-Asp-Xaa* (SEQ ID NO: 109) Proprotein convertase Arg-(Xaa)_(n)-Arg-Xaa*; 1 n = 0, 2, 4 or 6 (SEQ ID NO: 110)Proprotein convertase  Lys-(Xaa)_(n)-Arg-Xaa*; 2 n = 0, 2,4, or 6(SEQ ID NO: 111) Proprotein convertase  Glu-Arg-Thr-Lys-Arg-Xaa* 4(SEQ ID NO: 112) Proprotein convertase  Arg-Val-Arg-Arg-Xaa* 4 PACE 4(SEQ ID NO: 113) Decanoyl-Arg-Val-Arg-Arg- Xaa* (SEQ ID NO: 114)Prolyloligopeptidase  Pro-Xaa*-Trp-Val-Pro-Xaa Endothelin cleaving(SEQ ID NO: 115) enzyme in combination with dipeptidyl- peptidase IVSignal peptidase Trp-Val*-Ala-Xaa (SEQ ID NO: 116) Neprilysin in Xaa-Phe*-Xaa-Xaa combination with (SEQ ID NO: 117) dipeptidyl-peptidase Xaa-Tyr*-Xaa-Xaa IV (SEQ ID NO: 118) Xaa-Trp*-Xaa-Xaa (SEQ ID NO: 119)Renin in Asp-Arg-Tyr-Ile-Pro-Phe- combination withHis-Leu*-Leu (Val, Ala or  dipeptidyl-peptidase Pro)-Tyr-(Ser, Pro, or Ala) IV (SEQ ID NO: 120)

Thus, in various embodiments, the cleavable linker can be any cleavablelinker known by one of skill in the art to be cleavable by an enzymethat is present at the basolateral membrane of an epithelial cell. Invarious embodiments, the cleavable linker comprises a peptide. In otherembodiments, the cleavable linker comprises a nucleic acid, such as RNAor DNA. In still other embodiments, the cleavable linker comprises acarbohydrate, such as a disaccharide or a trisaccharide.

Alternatively, in various embodiments, the cleavable linker can be anycleavable linker known by one of skill in the art to be cleavable by anenzyme that is present in the plasma of the subject to whom the deliveryconstruct is administered. In various embodiments, the cleavable linkercomprises a peptide. In other embodiments, the cleavable linkercomprises a nucleic acid, such as RNA or DNA. In still otherembodiments, the cleavable linker comprises a carbohydrate, such as adisaccharide or a trisaccharide.

In various embodiments, the peptidases exhibit much higher (e.g., 100%,200%, or more increase in activity relative to the apical side) on thebaso-lateral side (also referred to as basolateral). Thus, in variousembodiments, the cleavable linker is cleavable by an enzyme thatexhibits 50% higher activity on the basolateral side of the membranethan on the apical side of the membrane. In various embodiments, thecleavable linker is cleavable by an enzyme that exhibits 100% higheractivity on the basolateral side of the membrane than on the apical sideof the membrane. In various embodiments, the cleavable linker iscleavable by an enzyme that exhibits 200% higher activity on thebasolateral side of the membrane than on the apical side of themembrane. In various embodiments, the cleavable linker is cleavable byan enzyme that exhibits 500% higher activity on the basolateral side ofthe membrane than on the apical side of the membrane. In variousembodiments, the cleavable linker is cleavable by an enzyme thatexhibits 1,000% higher activity on the basolateral side of the membranethan on the apical side of the membrane.

In various embodiments, the fusion molecule comprises a cleavable linkerhaving an amino acid sequence selected from, e.g., SEQ ID NO: 100, SEQID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106 or SEQ ID NO: 107 and is cleavable by an enzyme thatexhibits higher activity on the basolateral side of a polarizedepithelial cell than it does on the apical side of the polarizedepithelial cell, and/or is cleavable by an enzyme that exhibits higheractivity in the plasma than it does on the apical side of a polarizedepithelial cell.

In various embodiments, the cleavable linker can be a cleavable linkerthat is cleaved following a change in the environment of the fusionmolecule. For example, the cleavable linker can be a cleavable linkerthat is pH sensitive and is cleaved by a change in pH that isexperienced when the fusion molecule is released from the basolateralmembrane of a polarized epithelial cell. For instance, the intestinallumen is strongly alkaline, while plasma is essentially neutral. Thus, acleavable linker can be a moiety that is cleaved upon a shift fromalkaline to neutral pH. The change in the environment of the fusionmolecule that cleaves the cleavable linker can be any environmentalchange that that is experienced when the fusion molecule is releasedfrom the basolateral membrane of a polarized epithelial cell known byone of skill in the art, without limitation.

In various embodiments, the cleavable linker is cleaved by a cleavingenzyme found in the plasma of the subject. Any cleaving enzyme known byone of skill in the art to be present in the plasma of the subject canbe used to cleave the cleavable linker. Accordingly, in variousembodiments, the cleavable linker can be cleaved with an enzyme that isselected from e.g., SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO:115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 orSEQ ID NO: 120.

In various embodiment, the cleavable linker is a linker that contains anamino acid sequence that is a known substrate for the tobacco etch virus(TEV) protease. Accordingly, in various embodiments, the cleavablelinker comprises the amino acid sequence set in forth in, e.g.,GGGGSGGGENLYFQS (SEQ ID NO: 121).

Chemical Conjugation of the Cargo to the modified Cholix Toxin

In various embodiments, the biologically active cargo to be delivered tothe subject is chemically conjugated to the modified Cholix toxin. Meansof chemically conjugating molecules are well known to those of skill.

The procedure for conjugating two molecules varies according to thechemical structure of the agent. Polypeptides typically contain varietyof functional groups; e.g., carboxylic acid (COOH) or free amine (—NH₂)groups, that are available for reaction with a suitable functional groupon the other peptide, or on a linker to join the molecules thereto.

Alternatively, the antibody and/or the biologically active cargo can bederivatized to expose or attach additional reactive functional groups.The derivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

In various embodiments, isolated modified Cholix toxins are prepared bybacterial fermentation and purified by established methods. The purifiedmodified Cholix toxin is then modified at its C-terminus to allow directchemical coupling through a free sulfhydryl residue located near theC-terminus of the protein. The C-terminal modification includes acysteine-constrained loop harboring the consensus cleavage sequence forthe highly selective protease from the tobacco etch virus (TEV), asecond cysteine, and a hexa-histadine (His₆) tag. The second Cys isincluded to form a disulphide bridge with the Cys ultimately used forcoupling. Adding the His₆ sequence to the protein simplifies thepurification and the TEV cleavage sequence provides a mechanism toselectively remove the terminal Cys residue following mild reduction.TEV cleavage and mild reduction with 0.1 mM dithiotheitol followingexpression and isolation of the ntCholix constructs allows for thedirect chemical coupling of a biologically active cargo via amaleimide-based reaction as a generic mechanism of cargo attachment.Following TEV protease cleavage, reduction, and cargo coupling through amaleimide reaction with the free sulfhydryl, removal of the freedC-terminal sequence was achieved by a second Ni²⁺ column chromatographystep.

In various embodiments, the fusion molecule comprises particles whichare decorated covalently with the modified Cholix toxin, and wherein thebiologically active cargo is integrated into the particles. In variousembodiments, the particles can be smaller than ˜150 nm in diameter,smaller than ˜100 nm, or smaller than ˜50 nm.

In various embodiments, the fusion molecule comprises a biologicallyactive cargo coupled non-covalently to the modified Cholix toxin. Thisfusion molecule could ferry, e.g., a non-covalently associated IL-10across the epithelium such as a surface element of the IL-10 receptor(Josephson, K., Logsdon, N. J., Walter, M. R., Immunity 15: 35-46, 2001,incorporated by reference in its entirety herein).

Pharmaceutical Compositions and Delivery Methods

The pharmaceutical compositions of the present disclosure relate tocompositions for administration to a human subject. The pharmaceuticalcompositions comprise the non-naturally occurring fusion moleculesrecited herein, alone or in combination. The pharmaceutical compositionsmay comprise additional molecules capable of altering thecharacteristics of the non-naturally occurring fusion molecules, forexample, stabilizing, modulating and/or activating their function. Thecomposition may, e.g., be in solid or liquid form and may be, interalia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or(an) aerosol(s). The pharmaceutical composition of the presentdisclosure may, optionally and additionally, comprise a pharmaceuticallyacceptable carrier. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial and any of the standard pharmaceutical carriers, vehicles,buffers, and excipients, such as a phosphate buffered saline solution,5% aqueous solution of dextrose, and emulsions, such as an oil/water orwater/oil emulsion, and various types of wetting agents and/oradjuvants.

The pharmaceutical compositions are generally formulated appropriatelyfor the immediate use intended for the fusion molecule. For example, ifthe fusion molecule is not to be administered immediately, the fusionmolecule can be formulated in a composition suitable for storage. Onesuch composition is a lyophilized preparation of the fusion moleculetogether with a suitable stabilizer. Alternatively, the fusion moleculecomposition can be formulated for storage in a solution with one or moresuitable stabilizers. Any such stabilizer known to one of skill in theart without limitation can be used. For example, stabilizers suitablefor lyophilized preparations include, but are not limited to, sugars,salts, surfactants, proteins, chaotropic agents, lipids, and aminoacids. Stabilizers suitable for liquid preparations include, but are notlimited to, sugars, salts, surfactants, proteins, chaotropic agents,lipids, and amino acids. Specific stabilizers than can be used in thecompositions include, but are not limited to, trehalose, serum albumin,phosphatidylcholine, lecithin, and arginine. Other compounds,compositions, and methods for stabilizing a lyophilized or liquidpreparation of the fusion molecules may be found, for example, in U.S.Pat. Nos. 6,573,237, 6,525,102, 6,391,296, 6,255,284, 6,133,229,6,007,791, 5,997,856, and 5,917,021.

In various embodiments, the pharmaceutical compositions of the presentdisclosure are formulated for oral delivery. The pharmaceuticalcompositions formulated for oral administration take advantage of themodified Cholix toxin's ability to mediate transcytosis across thegastrointestinal (GI) epithelium. It is anticipated that oraladministration of these pharmaceutical compositions will result inabsorption of the fusion molecule through polarized epithelial cells ofthe digestive mucosa, e.g., the intestinal mucosa, followed by releaseof the biologically active cargo at the basolateral side of the mucousmembrane. In various embodiments, the epithelial cell is selected fromthe group consisting of nasal epithelial cells, oral epithelial cells,intestinal epithelial cells, rectal epithelial cells, vaginal epithelialcells, and pulmonary epithelial cells. Pharmaceutical compositions ofthe disclosure may include the addition of a transcytosis enhancer tofacilitate transfer of the fusion protein across the GI epithelium. Suchenhancers are known in the art. See Xia et al., (2000) J. Pharmacol.Experiment. Therap., 295:594-600; and Xia et al. (2001) PharmaceuticalRes., 18(2):191-195, each incorporated by reference in its entiretyherein.

It is anticipated that once transported across the GI epithelium, thefusion molecules of the disclosure will exhibit extended half-life inserum, that is, the biologically active cargo of the fusion moleculeswill exhibit an extended serum half-life compared to the biologicallyactive cargo in its non-fused state. As such, the oral formulations ofthe pharmaceutical compositions of the present disclosure are preparedso that they are suitable for transport to the GI epithelium andprotection of the fusion molecule in the stomach. Such formulations mayinclude carrier and dispersant components and may be in any suitableform, including aerosols (for oral or pulmonary delivery), syrups,elixirs, tablets, including chewable tablets, hard or soft capsules,troches, lozenges, aqueous or oily suspensions, emulsions, cachets orpellets granulates, and dispersible powders. In various embodiments, thepharmaceutical compositions are employed in solid dosage forms, e.g.,tablets, capsules, or the like, suitable for simple oral administrationof precise dosages.

In various embodiments, the oral formulation comprises a fusion moleculeand one or more compounds that can protect the fusion molecule while itis in the stomach. For example, the protective compound should be ableto prevent acid and/or enzymatic hydrolysis of the fusion molecule. Invarious embodiments, the oral formulation comprises a fusion moleculeand one or more compounds that can facilitate transit of the constructfrom the stomach to the small intestine. In various embodiments, the oneor more compounds that can protect the fusion molecule from degradationin the stomach can also facilitate transit of the construct from thestomach to the small intestine. For example, inclusion of sodiumbicarbonate can be useful for facilitating the rapid movement ofintra-gastric delivered materials from the stomach to the duodenum asdescribed in Mrsny et al., Vaccine 17:1425-1433, 1999. Other methods forformulating compositions so that the fusion molecules can pass throughthe stomach and contact polarized epithelial membranes in the smallintestine include, but are not limited to, enteric-coating technologiesas described in DeYoung, Int J Pancreatol, 5 Suppl:31-6, 1989 and themethods provided in U.S. Pat. Nos. 6,613,332, 6,174,529, 6,086,918,5,922,680, and 5,807,832, each incorporated by reference in its entiretyherein.

Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents inorder to provide a pharmaceutically elegant and palatable preparation.For example, to prepare orally deliverable tablets, the fusion moleculeis mixed with at least one pharmaceutical excipient, and the solidformulation is compressed to form a tablet according to known methods,for delivery to the gastrointestinal tract. The tablet composition istypically formulated with additives, e.g. a saccharide or cellulosecarrier, a binder such as starch paste or methyl cellulose, a filler, adisintegrator, or other additives typically usually used in themanufacture of medical preparations. To prepare orally deliverablecapsules, DHEA is mixed with at least one pharmaceutical excipient, andthe solid formulation is placed in a capsular container suitable fordelivery to the gastrointestinal tract. Compositions comprising fusionmolecules may be prepared as described generally in Remington'sPharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa.18042) at Chapter 89, which is herein incorporated by reference.

In various embodiments, the pharmaceutical compositions are formulatedas orally deliverable tablets containing fusion molecules in admixturewith non-toxic pharmaceutically acceptable excipients which are suitablefor manufacture of tablets. These excipients may be inert diluents, suchas calcium carbonate, sodium carbonate, lactose, calcium phosphate orsodium phosphate; granulating and disintegrating agents, for example,maize starch, gelatin or acacia, and lubricating agents, for example,magnesium stearate, stearic acid, or talc. The tablets may be uncoatedor they may be coated with known techniques to delay disintegration andabsorption in the gastrointestinal track and thereby provide a sustainedaction over a longer period of time. For example, a time delay materialsuch as glyceryl monostearate or glyceryl distearate alone or with a waxmay be employed.

In various embodiments, the pharmaceutical compositions are formulatedas hard gelatin capsules wherein the fusion molecule is mixed with aninert solid diluent, for example, calcium carbonate, calcium phosphate,or kaolin or as soft gelatin capsules wherein the fusion molecule ismixed with an aqueous or an oil medium, for example, arachis oil, peanutoil, liquid paraffin or olive oil.

In various embodiments, aqueous suspensions may contain a fusionmolecule in the admixture with excipients suitable for the manufactureof aqueous suspensions. Such excipients are suspending agents, forexample, sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia; dispersing or wetting agents may be anaturally occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for example,polyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example,heptadecylethyloxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyoxyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives for example,ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents such as sucroseor saccharin.

In various embodiments, oily suspensions may be formulated by suspendingthe fusion molecule in a vegetable oil, for example, arachis oil, oliveoil, sesame oil or coconut oil, or in a mineral oil such as liquidparaffin. The oil suspensions may contain a thickening agent, forexample, beeswax, hard paraffin or cetyl alcohol. Sweetening agents,such as those set forth above, and flavoring agents may be added toprovide a palatable oral preparation. These compositions may bepreserved by the addition of an antioxidant such as ascorbic acid.

In various embodiments, the pharmaceutical compositions may be in theform of oil-in-water emulsions. The oil phase may be a vegetable oil,for example, olive oil or arachis oil, or a mineral oil for example, gumacacia or gum tragacanth, naturally-occurring phosphotides, for examplesoybean lecithin, and esters or partial esters derived from fatty acidsand hexitol anhydrides, for example, sorbitan monooleate, andcondensation products of the same partial esters with ethylene oxide,for example, polyoxyethylene sorbitan monooleate. The emulsions may alsocontain sweetening and flavoring agents.

In various embodiments wherein the pharmaceutical composition is in theform of a tablet or capsule, the tablet or capsule is coated orencapsulated to protect the biologically active cargo from enzymaticaction in the stomach and to ensure that there is sufficientbiologically active cargo to be absorbed by the subject to produce aneffective response. Such coating or encapsulation methods include, e.g.,encapsulation in nanoparticles composed of polymers with a hydrophobicbackbone and hydrophilic branches as drug carriers, encapsulation inmicroparticles, insertion into liposomes in emulsions, and conjugationto other molecules. Examples of nanoparticles include mucoadhesivenanoparticles coated with chitosan and Carbopol (Takeuchi et al., Adv.Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles containing chargedcombination polyesters, poly(2-sulfobutyl-vinyl alcohol) andpoly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm.50(1):147-160, 2000).

Encapsulated or coated tablets can be used that release the biologicallyactive cargo in a layer-by-layer manner, thereby releasing biologicallyactive cargo over a pre-determined time frame while moving along thegastrointestinal tract. In addition, tablets comprising the biologicallyactive cargo can be placed within a larger tablet, thereby protectingthe inner tablet from environmental and processing conditions, such astemperature, chemical agents (e.g., solvents), pH, and moisture. Theouter tablet and coatings further serve to protect the biologicallyactive cargo in the gastric environment.

In various embodiments, pharmaceutical compositions may be formulatedfor oral delivery using polyester microspheres, zein microspheres,proteinoid microspheres, polycyanoacrylate microspheres, and lipid-basedsystems (see, for example, DiBase and Morrel, Oral Delivery ofMicroencapsulated Proteins, in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).

Surface active agents or surfactants promote absorption of polypeptidesthrough mucosal membrane or lining. Useful surface active agents orsurfactants include fatty acids and salts thereof, bile salts,phospholipid, or an alkyl saccharide. Examples of fatty acids and saltsthereof include sodium, potassium and lysine salts of caprylate (C₈),caprate (C₁₀), laurate (C₁₂) and myristate (C₁₄). Examples of bile saltsinclude cholic acid, chenodeoxycholic acid, glycocholic acid,taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholicacid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid,lithocholic acid, and ursodeoxycholic acid. Examples of phospholipidsinclude single-chain phospholipids, such as lysophosphatidylcholine,lysophosphatidylglycerol, lysophosphatidylethanolamine,lysophosphatidylinositol and lysophosphatidylserine; or double-chainphospholipids, such as diacylphosphatidylcholines,diacylphosphatidylglycerols, diacylphosphatidylethanolamines,diacylphosphatidylinositols and diacylphosphatidylserines. Examples ofalkyl saccharides include alkyl glucosides or alkyl maltosides, such asdecyl glucoside and dodecyl maltoside.

In another aspect, the present disclosure relates to methods of orallyadministering the pharmaceutical compositions of the disclosure. Withoutintending to be bound to any particular theory or mechanism of action,it is believed that oral administration of the fusion molecules resultsin absorption of the fusion molecule through polarized epithelial cellsof the digestive mucosa, e.g., the intestinal mucosa, followed bycleavage of the fusion molecule and release of the biologically activecargo at the basolateral side of the mucous membrane. Thus, when thebiologically active cargo exerts a biological activity in the liver,such as, for example, activities mediated by IL-10 binding to itscognate receptor, the biologically active cargo is believed to exert aneffect in excess of what would be expected based on the plasmaconcentrations observed in the subject, i.e., oral administration of thefusion molecule can deliver a higher effective concentration of thedelivered biologically active cargo to the liver of the subject than isobserved in the subject's plasma.

In another aspect, the present disclosure relates to methods of orallyadministering the pharmaceutical compositions of the disclosure. Suchmethods may include, but are not limited to, steps of orallyadministering the compositions by the patient or a caregiver. Suchadministration steps may include administration on intervals such asonce or twice per day depending on the fusion molecule, disease orpatient condition or individual patient. Such methods also include theadministration of various dosages of the individual fusion molecule. Forinstance, the initial dosage of a pharmaceutical composition may be at ahigher level to induce a desired effect, such as reduction in bloodglucose levels. Subsequent dosages may then be decreased once a desiredeffect is achieved. These changes or modifications to administrationprotocols may be done by the attending physician or health care worker.

These pharmaceutical compositions can be administered to the subject ata suitable dose. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. The therapeuticallyeffective amount for a given situation will readily be determined byroutine experimentation and is within the skills and judgment of theordinary clinician or physician. The skilled person knows that theeffective amount of a pharmaceutical composition administered to anindividual will, inter alia, depend on the nature of the biologicallyactive cargo. The length of treatment needed to observe changes and theinterval following treatment for responses to occur vary depending onthe desired effect. The particular amounts may be determined byconventional tests which are well known to the person skilled in theart.

The amount of biologically active cargo is an amount effective toaccomplish the purpose of the particular active agent. The amount in thecomposition typically is a pharmacologically, biologically,therapeutically, or chemically effective amount. However, the amount canbe less than a pharmacologically, biologically, therapeutically, orchemically effective amount when the composition is used in a dosageunit form, such as a capsule, a tablet or a liquid, because the dosageunit form may contain a multiplicity of carrier/biologically orchemically active agent compositions or may contain a dividedpharmacologically, biologically, therapeutically, or chemicallyeffective amount. The total effective amounts can then be administeredin cumulative units containing, in total, pharmacologically,biologically, therapeutically or chemically active amounts ofbiologically active cargo.

In various embodiments, an amount of fusion molecule administered to thesubject is at most 0.001 pg, at most 1 pg, at most 2 pg, at most 3 pg,at most 4 pg, at most 5 pg, at most 10 pg, at most 50 pg, at most 100pg, at most 1 μg, at most 2 μg, at most 3 μg, at most 4 μg, at most 5μg, at most 10 μg, at most 50 μg, at most 100 μg, at most 1 mg, at most2 mg, at most 3 mg, at most 4 mg, at most 5 mg, at most 10 mg, at most50 mg, at most 100 mg, or at most 1 g.

In various embodiments, an amount of fusion molecule administered to thesubject is at least 0.001 pg, at least 1 pg, at least 2 pg, at least 3pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 50 pg, atleast 100 pg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4μg, at least 5 μg, at least 10 μg, at least 50 μg, at least 100 μg, atleast 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg,at least 10 mg, at least 50 mg, at least 100 mg, or at least 1 g.

In various embodiments, an amount of fusion molecule administered to thesubject is from 0.001 pg and about 1 g, from1 pg to 10 pg, from 50 pg to100 pg, from1 μg to 5 μg, from 10 μg to 20 μg, from 10 μg to 500 mg,from 10 μg to 100 mg, from 10 μg to 1000 μg, from 10 μg to 250 μg, from10 μg to 100 μg, from 10 μg to 50 pg, from 1 mg to 5 mg, or from 10 mgto 100 mg.

The volume of a composition comprising the fusion molecule that isadministered will generally depend on the concentration of fusionmolecule and the formulation of the composition. In various embodiments,a unit dose of the fusion molecule composition is from 0.001 μl to 1 ml,from 1 μl to 100 μl, from 50 μl to 500 μl, from 0.01 ml to 1 ml, from 1ml to 100 ml, from 0.05 ml to 1 ml. For example, the unit dose of thefusion molecule composition can be about 0.5 ml.

In some embodiments, a unit dose of the fusion molecule composition isat most about 0.001 μl, at most 1 μl, at most 10 μl, at most 50 μl, atmost 200 μl, at most 0.01 ml, at most 0.05 ml, at most 0.1 ml, at most0.2 ml, at most 0.5 ml, or at most 1 ml.

In some a unit dose of the fusion molecule composition is at least 0.001μl, at least 1 μl, at least 10 μl, at least 50 μl, at least 200 μl, atleast 0.01 ml, at least 0.05 ml, at least 0.1 ml, at least 0.2 ml, atleast 0.5 ml, or at least 1 ml.

The fusion molecule compositions can be prepared in dosage formscontaining between 1 and 50 doses (e.g., 0.5 ml to 25 ml), more usuallybetween 1 and 10 doses (e.g., 0.5 ml to 5 ml).

The fusion molecule compositions of the disclosure can be administeredin one dose or in multiple doses. A dose can be followed by one or moredoses spaced by about 1 to about 6 hours, by about 6 to about 12 hours,by about 12 to about 24 hours, by about 1 day to about 3 days, by about1 day to about 1 week, by about 1 week to about 2 weeks, by about 2weeks to about 1 month, by about 4 to about 8 weeks, by about 1 to about3 months, or by about 1 to about 6 months.

In various embodiments, the pharmaceutical compositions comprising thefusion molecules may be, though not necessarily, administered daily, inan effective amount to ameliorate a symptom. Generally, the total dailydosage can be administered at an amount of at least about 0.001 pg, atleast about 0.1 mg, at least about 1 mg, at least about 10 mg, at leastabout 50 mg, at least about 100 mg, at least about 150 mg, at leastabout 200 mg, at least about 250 mg, at least about 300 mg, at leastabout 350 mg, at least about 400 mg, at least about 450 mg, at leastabout 500 mg per day, or at least about 1000 mg per day. For example,the dosage can be formulated for oral administration in capsules ortablets, such that 4 capsules or tablets, each containing 50 mg fusionmolecule. Capsules or tablets for oral delivery can conveniently containup to a full daily oral dose, e.g., 200 mg or more per day.

In various embodiments, the pharmaceutical compositions comprising thefusion molecules may be, though not necessarily, administered daily, inan effective amount to ameliorate a symptom. Generally, the total dailydosage can be administered at an amount of at most 50 mg per day, atmost 100 mg per day, at most 150 mg per day, at most 200 mg per day, atmost 250 mg per day, at most 300 mg per day, at most 350 mg per day, atmost 400 mg per day, at most 450 mg per day, at most 500 mg per day, orat most 1000 mg per day.

As used herein, the terms “co-administration”, “co-administered” and “incombination with”, referring to the fusion molecules of the disclosureand one or more other therapeutic agents, is intended to mean, and doesrefer to and include the following: simultaneous administration of suchcombination of fusion molecules of the disclosure and therapeuticagent(s) to a patient in need of treatment, when such components areformulated together into a single dosage form which releases saidcomponents at substantially the same time to said patient; substantiallysimultaneous administration of such combination of fusion molecules ofthe disclosure and therapeutic agent(s) to a patient in need oftreatment, when such components are formulated apart from each otherinto separate dosage forms which are taken at substantially the sametime by said patient, whereupon said components are released atsubstantially the same time to said patient; sequential administrationof such combination of fusion molecules of the disclosure andtherapeutic agent(s) to a patient in need of treatment, when suchcomponents are formulated apart from each other into separate dosageforms which are taken at consecutive times by said patient with asignificant time interval between each administration, whereupon saidcomponents are released at substantially different times to saidpatient; and sequential administration of such combination of fusionmolecules of the disclosure and therapeutic agent(s) to a patient inneed of treatment, when such components are formulated together into asingle dosage form which releases said components in a controlled mannerwhereupon they are released in a concurrent, consecutive, and/oroverlapping manner at the same and/or different times to said patient,where each part may be administered by either the same or a differentroute.

In various embodiments, the pharmaceutical compositions comprising thefusion molecules may be co-administered with a second component, whereinthe second component is a hormone, toxin, or bioactive agent which iscapable of binding to the GM-1 (monosialotetrahexosylganglioside)receptor (Hakomori, Advances in Exp. Medicine and Biology, 174:333-339,1984). In various embodiments, the second component is SV40 virus,polyoma virus, or a toxin such as cholera toxin, or exotoxin A fromPseudomonas aeruginosa (PE).

As used herein, the terms “cholera toxin” or “CT” refer to the eponymousvirulence agent of Vibrio cholerae bacterium, which can cause acute,life-threatening massive watery diarrhea. CT is a protein complexcomposed of a single A subunit organized with a pentamer of B subunitsthat binds to cell surface G_(M1) ganglioside structures at the apicalsurface of epithelia. CT is secreted by V. cholera following horizontalgene transfer with virulent strains of V. cholerae carrying a variant oflysogenic bacteriophage called CTXf or CTXφ. Recent cholera outbreaks,however, have suggested that strains of some serogroups (non-O1,non-O139) do not express CT but rather use other virulence factors.Detailed analyses of non-O1, non-O139 environmental and clinical datasuggested the presence of a novel putative secreted exotoxin with somesimilarity to PE. The sequence of CT is known and has been described(Mekalanos J. J. et al Nature 306, page 551 (1983)).

As used herein the terms “exotoxin A from Pseudomonas aeruginosa”,“Pseudomonas exotoxin A” or “PE” refer to an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The 613-residuesequence of PE is well known in the art and is set forth, for example,in U.S. Pat. No. 5,602,095. Domain la (amino acids 1-252) mediates cellbinding. Domain II (amino acids 253-364) is responsible fortranslocation into the cytosol and domain III (amino acids 400-613)mediates ADP ribosylation of elongation factor 2. The function of domainIb (amino acids 365-399) remains undefined, although it has been known alarge part of it, amino acids 365-380, can be deleted without loss ofcytotoxicity. See Siegall et al., J Biol Chem, 264:14256-61 (1989).

Certain cytotoxic fragments of PE are known in the art and are oftenreferenced by the molecular weight of the fragment, which designates forthe person of skill in the art the particular composition of the PEfragment. For example, PE40 was one of the first fragments that wasstudied and used as the toxic portion of immunotoxins. The termdesignates a truncated form of PE in which domain Ia, the domainresponsible for non-specific binding. See, e.g., Pai et al., Proc. Nat'lAcad. Sci. USA, 88:3358-3362 (1991); and Kondo et al., J. Biol. Chem.,263:9470-9475 (1988). Elimination of non-specific binding, however, canalso be achieved by mutating certain residues of domain Ia. U.S. Pat.No. 5,512,658, for instance, discloses that a mutated PE in which domainIa is present but in which the basic residues of domain Ia at positions57, 246, 247, and 249 are replaced with acidic residues (glutamic acid,or “E”)) exhibits greatly diminished non-specific cytotoxicity. Thismutant form of PE is sometimes referred to as “PE4E.”

In various embodiments, the combination therapy comprises administeringthe isolated fusion molecule composition and the second agentcomposition simultaneously, either in the same pharmaceuticalcomposition or in separate pharmaceutical compositions. In variousembodiments, isolated fusion molecule composition and the second agentcomposition are administered sequentially, i.e., the isolated fusionmolecule composition is administered either prior to or after theadministration of the second agent composition.

In various embodiments, the administrations of the isolated fusionmolecule composition and the second agent composition are concurrent,i.e., the administration period of the isolated fusion moleculecomposition and the second agent composition overlap with each other.

In various embodiments, the administrations of the isolated fusionmolecule composition and the second agent composition arenon-concurrent. For example, in various embodiments, the administrationof the isolated fusion molecule composition is terminated before thesecond agent composition is administered. In various embodiments, theadministration second agent composition is terminated before theisolated fusion molecule composition is administered. In variousembodiments, the administrations of the fusion molecule of theinvention, whether alone or in combination with a therapeutic agent, canbe archived with a meal, e.g. prior to the meal, during the meal orafter the meal.

In some embodiments, the administration of the fusion molecule of theinvention, whether alone or in combination with a therapeutic agent, canbe archived prior to a meal. In various embodiments, the fusion moleculeof the invention, whether alone or in combination with a therapeuticagent, can be administered more than 12 hours, more than 11 hours, morethan 10 hours, more than 9 hours, more than 8 hours, more than 7 hours,more than 6 hours, more than 5 hours, more than 4 hours, more than 3hours, more than 2 hours, more than 1 hour, more than 50 minutes, morethan 40 minutes, more than 30 minutes, more than 20 minutes, more than10 minutes, more than 5 minutes, or more than 1 minute prior to themeal. In various embodiments, the fusion molecule of the invention,whether alone or in combination with a therapeutic agent, can beadministered less than 12 hours, less than 11 hours, less than 10 hours,less than 9 hours, less than 8 hours, less than 7 hours, less than 6hours, less than 5 hours, less than 4 hours, less than 3 hours, lessthan 2 hours, less than 1 hour, less than 50 minutes, less than 40minutes, less than 30 minutes, less than 20 minutes, less than 10minutes, less than 5 minutes, or less than 1 minute prior to the meal.In various embodiments, the fusion molecule of the invention, whetheralone or in combination with a therapeutic agent, can be administeredbetween about 1 minute to about 10 minutes, between about 5 minutes toabout 30 minutes, between about 20 minutes to about 60 minutes, betweenabout 1 hour to about 3 hours, between about 2 hours to about 10 hours,or between about 5 hours to about 12 hour prior to the meal.

In some embodiments, the administration of the fusion molecule of theinvention, whether alone or in combination with a therapeutic agent, canbe archived after a meal. In various embodiments, the fusion molecule ofthe invention, whether alone or in combination with a therapeutic agent,can be administered more than 12 hours, more than 11 hours, more than 10hours, more than 9 hours, more than 8 hours, more than 7 hours, morethan 6 hours, more than 5 hours, more than 4 hours, more than 3 hours,more than 2 hours, more than 1 hour, more than 50 minutes, more than 40minutes, more than 30 minutes, more than 20 minutes, more than 10minutes, more than 5 minutes, or more than 1 minute after the meal. Insome embodiments, the fusion molecule of the invention, whether alone orin combination with a therapeutic agent, can be administered less than12 hours, less than 11 hours, less than 10 hours, less than 9 hours,less than 8 hours, less than 7 hours, less than 6 hours, less than 5hours, less than 4 hours, less than 3 hours, less than 2 hours, lessthan 1 hour, less than 50 minutes, less than 40 minutes, less than 30minutes, less than 20 minutes, less than 10 minutes, less than 5minutes, or less than 1 minute after the meal. In various embodiments,the fusion molecule of the invention, whether alone or in combinationwith a therapeutic agent, can be administered less than 12 hours, lessthan 11 hours, less than 10 hours, less than 9 hours, less than 8 hours,less than 7 hours, less than 6 hours, less than 5 hours, less than 4hours, less than 3 hours, less than 2 hours, less than 1 hour, less than50 minutes, less than 40 minutes, less than 30 minutes, less than 20minutes, less than 10 minutes, less than 5 minutes, or less than 1minute prior to the meal. In various embodiments, the fusion molecule ofthe invention, whether alone or in combination with a therapeutic agent,can be administered between about 1 minute to about 10 minutes, betweenabout 5 minutes to about 30 minutes, between about 20 minutes to about60 minutes, between about 1 hour to about 3 hours, between about 2 hoursto about 10 hours, or between about 5 hours to about 12 hour after themeal.

Methods of Use

In another aspect, the pharmaceutical compositions formulated for oraldelivery are used to treat certain classes of diseases or medicalconditions that are particularly amenable for oral formulation anddelivery. Such classes of diseases or conditions include, e.g., viraldisease or infections, cancer, a metabolic diseases, obesity, autoimmunediseases, inflammatory diseases, allergy, graft-vs-host disease,systemic microbial infection, anemia, cardiovascular disease, psychosis,genetic diseases, neurodegenerative diseases, disorders of hematopoieticcells, diseases of the endocrine system or reproductive systems,gastrointestinal diseases. In many chronic diseases, oral formulationsof the fusion molecules of the disclosure are particularly usefulbecause they allow long-term patient care and therapy via home oraladministration without reliance on injectable treatment or drugprotocols.

In various embodiments of the present disclosure, pharmaceuticalcompositions comprising the fusion molecules of the disclosure areprovided for use in treating and/or preventing inflammatory diseases.“Inflammatory diseases” include all diseases associated with acute orchronic inflammation. Acute inflammation is the initial response of thebody to harmful stimuli and results from an increased movement of plasmaand leukocytes (such as e.g. granulocytes) from the blood into theinjured tissues. A number of biochemical events propagates and maturesthe inflammatory response, involving the local vascular system, theimmune system, and various cells within the injured tissue. Prolongedinflammation is referred to as chronic inflammation, which leads to aprogressive shift in the type of cells present at the site ofinflammation and is characterized by simultaneous destruction andhealing of the tissue from the inflammatory process. Inflammatorydiseases can be caused by e.g. burns, chemical irritants, frostbite,toxins, infection by pathogens, physical injury, immune reactions due tohypersensitivity, ionizing radiation, or foreign bodies, such as e.g.splinters, dirt and debris. Examples of inflammatory diseases are wellknown in the art.

In various embodiments, the inflammatory disease is selected from thegroup consisting of inflammatory bowel disease, psoriasis and bacterialsepsis. The term “inflammatory bowel disease”, as used herein, refers toa group of inflammatory conditions of the colon and small intestineincluding, for example, Crohn's disease, ulcerative colitis, collagenouscolitis, lymphocytic colitis, ischaemic colitis, diversion colitis,Behcet's syndrome and indeterminate colitis.

“Crohn's disease”, in accordance with the present disclosure, is aT-helper Type 1 (Th 1) inflammatory bowel disease, which has an immuneresponse pattern that includes an increased production ofinterleukin-12, tumour necrosis factor (TNF), and interferon-γ(Romagnani. Inflamm Bowel Dis 1999; 5:285-94), and which can have adevastating impact on the lifestyle of a patient afflicted therewith.Common symptoms of Crohn's disease include diarrhea, cramping, abdominalpain, fever, and even rectal bleeding. Crohn's disease and complicationsassociated with it often results in the patient requiring surgery, oftenmore than once. There is no known cure for Crohn's disease, andlong-term, effective treatment options are limited. The goals oftreatment are to control inflammation, correct nutritional deficiencies,and relieve symptoms like abdominal pain, diarrhea, and rectal tobleeding. While treatment can help control the disease by lowering thenumber of times a person experiences a recurrence, there is no cure.Treatment may include drugs, nutrition supplements, surgery, or acombination of these options. Common treatments which may beadministered for treatment include anti-inflammation drugs, includingsulfasalazine, cortisone or steroids, including prednisone, immunesystem suppressors, such as 6-mercaptopurine or azathioprine, andantibiotics.

“Psoriasis”, in accordance with the present disclosure, is a diseasewhich affects the skin and joints. It commonly causes red scaly patchesto appear on the skin. The scaly patches caused by psoriasis, calledpsoriatic plaques, are areas of inflammation and excessive skinproduction. Skin rapidly accumulates at these sites and takes asilvery-white appearance. Plaques frequently occur on the skin of theelbows and knees, but can affect any area including the scalp andgenitals. Psoriasis is hypothesized to be immune-mediated and is notcontagious. The disorder is a chronic recurring condition which variesin severity from minor localised patches to complete body coverage.Fingernails and toenails are frequently affected (psoriatic naildystrophy)—and can be seen as an isolated finding. Psoriasis can alsocause inflammation of the joints, which is known as psoriatic arthritis.Ten to fifteen percent of people with psoriasis have psoriaticarthritis.

The term “bacterial sepsis”, as used herein, refers to life-threateningconditions resulting from the circulation of bacteria in the bloodstream. Sepsis results in generalized systemic production ofproinflammatory cytokines that results in tissue damage and ultimatelyseptic shock due to failure of the microcirculation.

Another aspect of the present disclosure relates to methods fortreatment, prophylaxis and/or prevention of an autoimmune disease,comprising administering to said patient a therapeutically effectiveamount (either as monotherapy or in a combination therapy regimen) of afusion molecule described herein, in pharmaceutically acceptablecarrier.

An autoimmune disease, as pertains to the present disclosure, is adisease or disorder arising from and directed against an individual'sown tissues or a co-segregate or manifestation thereof or resultingcondition therefrom. In various embodiments the autoimmune disease isselected from the group consisting of systemic lupus erythematosus(SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia,thrombocytopenia purpura, Grave's disease, Sjogren's disease,dermatomyositis, Hashimoto's disease, polymyositis, inflammatory boweldisease, multiple sclerosis (MS), diabetes mellitus, rheumatoidarthritis, and scleroderma.

“Rheumatoid arthritis”, in accordance with the present disclosure, is anautoimmune disorder that causes the body's immune system to attack thebone joints (Muller B et al., Springer Semin Immunopathol., 20:181-96,1998). Rheumatoid arthritis is a chronic, systemic inflammatory disorderthat may affect many tissues and organs, but principally attackssynovial joints. The process produces an inflammatory response of thesynovium (synovitis) secondary to hyperplasia of synovial cells, excesssynovial fluid, and the development of pannus in the synovium. Thepathology of the disease process often leads to the destruction ofarticular cartilage and ankylosis of the joints. Rheumatoid arthritiscan also produce diffuse inflammation in the lungs, pericardium, pleura,and sclera, and also nodular lesions, most common in subcutaneous tissueunder the skin.

In various embodiments of the present disclosure, pharmaceuticalcompositions comprising the fusion molecules of the disclosure areprovided for use in the treatment, prophylaxis and/or prevention of acancer, comprising administering to said patient a therapeuticallyeffective amount (either as monotherapy or in a combination therapyregimen) of a fusion molecule described herein, in pharmaceuticallyacceptable carrier. Cancers to be treated include, but are not limitedto, non-Hodgkin's lymphomas, Hodgkin's lymphoma, chronic lymphocyticleukemia, hairy cell leukemia, acute lymphoblastic leukemia, multiplemyeloma, carcinomas of the pancreas, colon, gastric intestine, prostate,bladder, kidney ovary, cervix, breast, lung, nasopharynx, malignantmelanoma and rituximab resistant NHL and leukemia.

In various embodiments, the therapeutically effective amount of a fusionmolecule described herein will be administered in combination with oneor more other therapeutic agents. Such therapeutic agents may beaccepted in the art as a standard treatment for a particular diseasestate as described herein, such as inflammatory disease, autoimmunedisease, or cancer. Exemplary therapeutic agents contemplated include,but are not limited to, cytokines, growth factors, steroids, NSAIDs,DMARDs, anti-inflammatories, chemotherapeutics, radiotherapeutics, orother active and ancillary agents.

In various embodiments, the present disclosure provides a method oftreating a subject having a metabolic disorder, said method comprisingorally administering a fusion molecule of the present disclosure in anamount sufficient to treat said disorder, wherein said metabolicdisorder is diabetes, obesity, diabetes as a consequence of obesity,hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulinresistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, orhyperlipidemia.

In another aspect, the present disclosure provides a method of treatinga subject having a fatty liver disease (e.g., nonalcoholic fatty liverdisease (NAFLD); nonalcoholic steatohepatitis (NASH)), agastrointestinal disease, or a neurodegenerative disease, said methodcomprising orally administering a fusion molecule of the presentdisclosure in an amount sufficient to treat said disease.

In another aspect, the present disclosure relates to the use of anon-naturally occurring fusion molecule of the present disclosure forthe preparation of a medicament for treatment, prophylaxis and/orprevention of GH deficient growth disorders in a subject in needthereof.

In another aspect, the present disclosure provides a method of treatinga subject having a GH deficient growth disorder, said method comprisingorally administering a fusion molecule of the present disclosure in anamount sufficient to treat said disorder, wherein said disorder isgrowth hormone deficiency (GHD), Turner syndrome (TS), Noonan syndrome,Prader-Willi syndrome, short stature homeobox-containing gene (SHOX)deficiency, chronic renal insufficiency, and idiopathic short statureshort bowel syndrome, GH deficiency due to rare pituitary tumors ortheir treatment, and muscle-wasting disease associated with HIV/AIDS.

Polynucleotides Encoding Fusion Molecules

In another aspect, the disclosure provides polynucleotides comprising anucleotide sequence encoding the non-naturally occurring fusionmolecules. These polynucleotides are useful, for example, for making thefusion molecules. In yet another aspect, the disclosure provides anexpression system that comprises a recombinant polynucleotide sequenceencoding a modified Cholix toxin, and a polylinker insertion site for apolynucleotide sequence encoding a biologically active cargo. Thepolylinker insertion site can be anywhere in the polynucleotide sequenceso long as the polylinker insertion does not disrupt the receptorbinding domain or the transcytosis domain of the modified Cholix toxin.In various embodiments, the expression system may comprise apolynucleotide sequence that encodes a cleavable linker so that cleavageat the cleavable linker separates a biologically active cargo encoded bya nucleic acid inserted into the polylinker insertion site from theremainder of the encoded fusion molecule. Thus, in embodiments where thepolylinker insertion site is at an end of the encoded construct, thepolynucleotide comprises one nucleotide sequence encoding a cleavablelinker between the polylinker insertion site and the remainder of thepolynucleotide. In embodiments where the polylinker insertion site isnot at the end of the encoded construct, the polylinker insertion sitecan be flanked by nucleotide sequences that each encode a cleavablelinker.

Various in vitro methods that can be used to prepare a polynucleotideencoding a modified Cholix toxin useful in the fusion molecules of thedisclosure include, but are not limited to, reverse transcription, thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (3SR) and the QP replicase amplificationsystem (QB). Any such technique known by one of skill in the art to beuseful in construction of recombinant nucleic acids can be used. Forexample, a polynucleotide encoding the protein or a portion thereof canbe isolated by polymerase chain reaction of cDNA using primers based onthe DNA sequence of a modified Cholix toxin or a nucleotide encoding,e.g., a receptor binding domain.

Guidance for using these cloning and in vitro amplificationmethodologies are described in, for example, U.S. Pat. No. 4,683,195;Mullis et al., 1987, Cold Spring Harbor Symp. Quant. Biol. 51:263; andErlich, ed., 1989, PCR Technology, Stockton Press, NY. Polynucleotidesencoding a fusion molecule or a portion thereof also can be isolated byscreening genomic or cDNA libraries with probes selected from thesequences of the desired polynucleotide under stringent, moderatelystringent, or highly stringent hybridization conditions.

Construction of nucleic acids encoding the fusion molecules of thedisclosure can be facilitated by introducing an insertion site for anucleic acid encoding the biologically active cargo into the construct.In various embodiments, an insertion site for the biologically activecargo can be introduced between the nucleotides encoding the cysteineresidues of domain Ib of the modified Cholix toxin. In otherembodiments, the insertion site can be introduced anywhere in thenucleic acid encoding the construct so long as the insertion does notdisrupt the functional domains encoded thereby. In various embodiments,the insertion site can be in the ER retention domain.

Further, the polynucleotides can also encode a secretory sequence at theamino terminus of the encoded fusion molecule. Such constructs areuseful for producing the fusion molecules in mammalian cells as theysimplify isolation of the immunogen.

Furthermore, the polynucleotides of the disclosure also encompassderivative versions of polynucleotides encoding a fusion molecule. Suchderivatives can be made by any method known by one of skill in the artwithout limitation. For example, derivatives can be made bysite-specific mutagenesis, including substitution, insertion, ordeletion of one, two, three, five, ten or more nucleotides, ofpolynucleotides encoding the fusion molecule. Alternatively, derivativescan be made by random mutagenesis. One method for randomly mutagenizinga nucleic acid comprises amplifying the nucleic acid in a PCR reactionin the presence of 0.1 mM MnCl₂ and unbalanced nucleotideconcentrations. These conditions increase the inaccuracy incorporationrate of the polymerase used in the PCR reaction and result in randommutagenesis of the amplified nucleic acid.

Accordingly, in various embodiments, the disclosure provides apolynucleotide that encodes a fusion molecule. The fusion moleculecomprises a modified Cholix toxin and a biologically active cargo to bedelivered to a subject; and, optionally, a non-cleavable or cleavablelinker. Cleavage at the cleavable linker can separate the biologicallyactive cargo from the remainder of the fusion molecule. The cleavablelinker can be cleaved by an enzyme that is present at a basolateralmembrane of a polarized epithelial cell of the subject or in the plasmaof the subject.

In various embodiments, the polynucleotide hybridizes under stringenthybridization conditions to any polynucleotide of this disclosure. Infurther embodiments, the polynucleotide hybridizes under stringentconditions to a nucleic acid that encodes any fusion molecule of thedisclosure.

In still another aspect, the disclosure provides expression vectors forexpressing the fusion molecules. Generally, expression vectors arerecombinant polynucleotide molecules comprising expression controlsequences operatively linked to a nucleotide sequence encoding apolypeptide. Expression vectors can readily be adapted for function inprokaryotes or eukaryotes by inclusion of appropriate promoters,replication sequences, selectable markers, etc. to result in stabletranscription and translation or mRNA. Techniques for construction ofexpression vectors and expression of genes in cells comprising theexpression vectors are well known in the art. See, e.g., Sambrook etal., 2001, Molecular Cloning—A Laboratory Manual, 3rd edition, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., and Ausubel et al.,eds., Current Edition, Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, NY.

Useful promoters for use in expression vectors include, but are notlimited to, a metallothionein promoter, a constitutive adenovirus majorlate promoter, a dexamethasone-inducible MMTV promoter, a SV40 promoter,a MRP pol III promoter, a constitutive MPSV promoter, atetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and a constitutive CMV promoter.

The expression vectors should contain expression and replication signalscompatible with the cell in which the fusion molecules are expressed.Expression vectors useful for expressing fusion molecules include viralvectors such as retroviruses, adenoviruses and adeno-associated viruses,plasmid vectors, cosmids, and the like. Viral and plasmid vectors arepreferred for transfecting the expression vectors into mammalian cells.For example, the expression vector pcDNA1 (Invitrogen, San Diego,Calif.), in which the expression control sequence comprises the CMVpromoter, provides good rates of transfection and expression into suchcells.

The expression vectors can be introduced into the cell for expression ofthe fusion molecules by any method known to one of skill in the artwithout limitation. Such methods include, but are not limited to, e.g.,direct uptake of the molecule by a cell from solution; facilitateduptake through lipofection using, e.g., liposomes or immunoliposomes;particle-mediated transfection; etc. See, e.g., U.S. Pat. No. 5,272,065;Goeddel et al., eds, 1990, Methods in Enzymology, vol. 185, AcademicPress, Inc., CA; Krieger, 1990, Gene Transfer and Expression—ALaboratory Manual, Stockton Press, NY; Sambrook et al., 1989, MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, NY; andAusubel et al., eds., Current Edition, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

The expression vectors can also contain a purification moiety thatsimplifies isolation of the fusion molecule. For example, apolyhistidine moiety of, e.g., six histidine residues, can beincorporated at the amino terminal end of the protein. The polyhistidinemoiety allows convenient isolation of the protein in a single step bynickel-chelate chromatography. In various embodiments, the purificationmoiety can be cleaved from the remainder of the fusion moleculefollowing purification. In other embodiments, the moiety does notinterfere with the function of the functional domains of the fusionmolecule and thus need not be cleaved.

In yet another aspect, the disclosure provides a cell comprising anexpression vector for expression of the fusion molecules, or portionsthereof. The cell is selected for its ability to express highconcentrations of the fusion molecule to facilitate purification of theprotein. In various embodiments, the cell is a prokaryotic cell, forexample, E. coli. As described in the examples, the fusion molecules areproperly folded and comprise the appropriate disulfide linkages whenexpressed in E. coli.

In other embodiments, the cell is a eukaryotic cell. Useful eukaryoticcells include yeast and mammalian cells. Any mammalian cell known by oneof skill in the art to be useful for expressing a recombinantpolypeptide, without limitation, can be used to express the fusionmolecules. For example, Chinese hamster ovary (CHO) cells can be used toexpress the fusion molecules.

The fusion molecules of the disclosure can be produced by recombination,as described below. However, the fusion molecules may also be producedby chemical synthesis using methods known to those of skill in the art.

Methods for expressing and purifying the fusion molecules of thedisclosure are described extensively in the examples below. Generally,the methods rely on introduction of an expression vector encoding thefusion molecule to a cell that can express the fusion molecule from thevector. The fusion molecule can then be purified for administration to asubject.

Transcytosis Testing

The function of the transcytosis domain can be tested as a function ofthe fusion molecule's ability to pass through an epithelial membrane.Because transcytosis first requires binding to the cell, these assayscan also be used to assess the function of the cell recognition domain.

The fusion molecule's transcytosis activity can be tested by any methodknown by one of skill in the art, without limitation. In variousembodiments, transcytosis activity can be tested by assessing theability of a fusion molecule to enter a non-polarized cell to which itbinds. Without intending to be bound to any particular theory ormechanism of action, it is believed that the same property that allows atranscytosis domain to pass through a polarized epithelial cell alsoallows molecules bearing the transcytosis domain to enter non-polarizedcells. Thus, the fusion molecule's ability to enter the cell can beassessed, for example, by detecting the physical presence of theconstruct in the interior of the cell. For example, the fusion moleculecan be labeled with, for example, a fluorescent marker, and the fusionmolecule exposed to the cell. Then, the cells can be washed, removingany fusion molecule that has not entered the cell, and the amount oflabel remaining determined. Detecting the label in this tractionindicates that the fusion molecule has entered the cell.

In other embodiments, the fusion molecule's transcytosis ability can betested by assessing the fusion molecule's ability to pass through apolarized epithelial cell. For example, the fusion molecule can belabeled with, for example, a fluorescent marker and contacted to theapical membranes of a layer of epithelial cells. Fluorescence detectedon the basolateral side of the membrane formed by the epithelial cellsindicates that the transcytosis domain is functioning properly.

Cleavable Linker Cleavage Testing

The function of the cleavable linker can generally be tested in acleavage assay. Any suitable cleavage assay known by one of skill in theart, without limitation, can be used to test the cleavable linkers. Bothcell-based and cell-free assays can be used to test the ability of anenzyme to cleave the cleavable linkers.

An exemplary cell-free assay for testing cleavage of cleavable linkerscomprises preparing extracts of polarized epithelial cells and exposinga labeled fusion molecule bearing a cleavable linker to the fraction ofthe extract that corresponds to membrane-associated enzymes. In suchassays, the label can be attached to either the biologically activecargo to be delivered or to the remainder of the fusion molecule. Amongthese enzymes are cleavage enzymes found near the basolateral membraneof a polarized epithelial cell, as described above. Cleavage can bedetected, for example, by binding the fusion molecule with, for example,an antibody and washing off unbound molecules. If label is attached tothe biologically active cargo to be delivered, then little or no labelshould be observed on the molecule bound to the antibodies.Alternatively, the binding agent used in the assay can be specific forthe biologically active cargo, and the remainder of the construct can belabeled. In either case, cleavage can be assessed.

Cleavage can also be tested using cell-based assays that test cleavageby polarized epithelial cells assembled into membranes. For example, alabeled fusion molecule, or portion of a fusion molecule comprising thecleavable linker, can be contacted to either the apical or basolateralside of a monolayer of suitable epithelial cells, such as, for example,Coco-2 cells, under conditions that permit cleavage of the linker.Cleavage can be detected by detecting the presence or absence of thelabel using a reagent that specifically binds the fusion molecule, orportion thereof. For example, an antibody specific for the fusionmolecule can be used to bind a fusion molecule comprising a label distalto the cleavable linker in relation to the portion of the fusionmolecule bound by the antibody. Cleavage can then be assessed bydetecting the presence of the label on molecules bound to the antibody.If cleavage has occurred, little or no label should be observed on themolecules bound to the antibody. By performing such experiments, enzymesthat preferentially cleave at the basolateral membrane rather than theapical membrane can be identified, and, further, the ability of suchenzymes to cleave the cleavable linker in a fusion molecule can beconfirmed.

Further, cleavage can also be tested using a fluorescence reporter assayas described in U.S. Pat. No. 6,759,207. Briefly, in such assays, thefluorescence reporter is contacted to the basolateral side of amonolayer of suitable epithelial cells under conditions that allow thecleaving enzyme to cleave the reporter. Cleavage of the reporter changesthe structure of the fluorescence reporter, changing it from anon-fluorescent configuration to a fluorescent configuration. The amountof fluorescence observed indicates the activity of the cleaving enzymepresent at the basolateral membrane.

Further, cleavage can also be tested using an intra-molecularly quenchedmolecular probe, such as those described in U.S. Pat. No. 6,592,847.Such probes generally comprise a fluorescent moiety that emits photonswhen excited with light of appropriate wavelength and a quencher moietythat absorbs such photons when in close proximity to the fluorescentmoiety. Cleavage of the probe separates the quenching moiety from thefluorescent moiety, such that fluorescence can be detected, therebyindicating that cleavage has occurred. Thus, such probes can be used toidentify and assess cleavage by particular cleaving enzymes bycontacting the basolateral side of a monolayer of suitable epithelialcells with the probe under conditions that allow the cleaving enzyme tocleave the probe. The amount of fluorescence observed indicates theactivity of the cleaving enzyme being tested.

Exemplary Cholix Toxin-Biologically Active Cargo Fusion Molecules

Embodiments of the present disclosure include, but are not limited to,the fusion molecules described in Table 7.

TABLE 7 Modified Cholix Biologically Active Toxin Cleavable Linker Cargo(SEQ ID NO) (SEQ ID NO) (SEQ ID NO) SEQ ID NO: 3 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 4 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 5 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 6 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 7 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 8 SEQID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 9 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 10 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 11 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 12 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 13 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 14 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 15SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 16 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 17 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 18 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 19 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 20 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 21 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 22SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 23 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 24 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 25 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 26 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 27 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 28 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 29SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 30 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 31 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 32 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 33 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 34 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 35 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 36SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 37 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 38 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 39 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 40 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 41 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 42 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 43SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 44 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 45 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 46 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 47 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 48 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 49 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 50SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 51 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 52 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 53 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 54 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 55 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 56 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 57SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 58 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 59 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 60 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 61 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 62 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 63 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 64SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 65 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 66 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 67 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 68 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 69 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 70 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 71SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 72 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 73 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 74 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker SEQ ID NO: 75 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 NoLinker SEQ ID NO: 76 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQID NO: 77 SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 78SEQ ID NOs: 96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 79 SEQ ID NOs:96-121 SEQ ID NOs: 82-95 No Linker SEQ ID NO: 80 SEQ ID NOs: 96-121 SEQID NOs: 82-95 No Linker SEQ ID NO: 81 SEQ ID NOs: 96-121 SEQ ID NOs:82-95 No Linker

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 82.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 82.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 82.

In various embodiments, the fusion molecule comprises the amino acidsequence set forth in SEQ ID NO: 114.

In various embodiments, the fusion molecule comprises the amino acidsequence set forth in SEQ ID NO: 115.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 88 and a light chainvariable having the amino acid sequence of SEQ ID NO: 89.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 90 and a light chainvariable having the amino acid sequence of SEQ ID NO: 91

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 92.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 93.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 94.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 52 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 95.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 88 and a light chainvariable having the amino acid sequence of SEQ ID NO: 89.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 90 and a light chainvariable having the amino acid sequence of SEQ ID NO: 91

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 92.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 93.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 94. In variousembodiments, the fusion molecule comprises a modified Cholix toxinhaving the amino acid sequence of SEQ ID NO: 80 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 95.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 88 and a light chainvariable having the amino acid sequence of SEQ ID NO: 89.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 90 and a light chainvariable having the amino acid sequence of SEQ ID NO: 91

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 92.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 93.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 94.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 70 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 95.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 88 and a light chainvariable having the amino acid sequence of SEQ ID NO: 89.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo that is an antibody comprising a heavy chain variablehaving the amino acid sequence of SEQ ID NO: 90 and a light chainvariable having the amino acid sequence of SEQ ID NO: 91.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 92.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 93.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 94.

In various embodiments, the fusion molecule comprises a modified Cholixtoxin having the amino acid sequence of SEQ ID NO: 42 and a biologicallyactive cargo having the amino acid sequence of SEQ ID NO: 95.

The following examples merely illustrate the disclosure, and are notintended to limit the disclosure in any way.

EXAMPLE 1

A plasmid construct was prepared encoding mature Vibrio cholera Cholixand used to express the mature Cholix protein in an E. coli expressionsystem as previously described; see, e.g., Jorgensen, R. et al., J BiolChem, 283(16):10671-10678 (2008). A non-toxic mutant form of the Cholixgene (hereinafter referred to as “ntCholix”) was also prepared bygenetic deletion of a glutamic acid at amino acid position 581 (ΔE581)which is analogous to a deletion (ΔE553) in the PE protein that rendersit non-toxic without significantly altering its conformation; Killeen,K. P. and Collier, R. J., Biochim Biophys Acta, 1138:162-166 (1992).Protein expression was achieved using E. coli DH5α cells (Invitrogen,Carlsbad, Calif.) following transformation by heat-shock (1 min at 42°C.) with the appropriate plasmid. Transformed cells, selected onantibiotic-containing media, were isolated and grown in Luria-Bertanibroth (Difco). Protein expression was induced by addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTGinduction, cells were harvested by centrifugation at 5,000×g for 10 minat 4° C. Inclusion bodies were isolated following cell lysis andproteins were solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0)plus 65 mM dithiothreitol. Following refolding and purification,proteins were stored at ˜5 ml/ml in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺at −80° C. All proteins used in these studies were confirmed to beat >90% purity based upon size exclusion chromatography.

The ntCholix form was then modified at its C-terminus to allow directchemical coupling through a free sulfhydryl residue located near theC-terminus of the protein. The strategy for the C-terminal modifcationis depicted in FIG. 1. The C-terminal modification included acysteine-constrained loop harboring the consensus cleavage sequence(ENLFQS) for the highly selective protease from the tobacco etch virus(TEV), a second cysteine, and a hexa-histadine (His₆) tag. The secondCys was included to form a disulphide bridge with the Cys ultimatelyused for coupling. Adding the His₆ sequence to the protein simplifiedthe purification and the TEV cleavage sequence provided a mechanism toselectively remove the terminal Cys residue following mild reduction.TEV cleavage and mild reduction with 0.1 mM dithiotheitol followingexpression and isolation of the ntCholix constructs allowed for thedirect chemical coupling of a cargo, Alexa Fluor® 488 fluorescent dye,via a maleimide-based reaction as a generic mechanism of cargoattachment. The resultant construct is referred to herein asntCholix-Alexa488. Following TEV protease cleavage, reduction, and cargocoupling through a maleimide reaction with the free sulfhydryl, removalof the freed C-terminal sequence was achieved by a second Ni²⁺ columnchromatography step.

EXAMPLE 2

Trans-epithelial transport of ntCholix-Alexa488 was assessed usingCaco-2 monolayers in vitro. First, Caco-2 cells (passage number 25-35)were grown to confluent monolayers as previously described; Rubas, W. etal., Pharm Res, 10:113-118 (1993). Briefly, cells were maintained at 37°C. in DMEM/high growth media enriched with 2 mM L-glutamine, 10% fetalbovine serum, and 100 Units of penicillin/streptomycin in an atmosphereof 5% CO₂ and 90% humidity. Cells were passaged every week at a splitratio of 1:3 in 75cm² flasks and seeded onto prewetted andcollagen-coated permeable (0.4 μm pore size) polycarbonate (Transwell™)filter supports from Corning Costar (Cambridge, Mass.) at a density of63,000 cells/cm². Growth media was replaced every other day. Confluentmonolayers, determined by the acquisition of significanttrans-epithelial resistance (TEER) determine using an volt-ohm-meter(World Precision Instruments, Sarasota, Fla.), were used 20-26 days postseeding.

Two additional materials were also prepared to be used as controls toassess the in vitro transport of Cholix. As an internal control forfilter damage, tetramethylrhodamine isothiocyanate (TRITC)-labeled 70kDa dextan was obtained from commercial source (Sigma). As a control fornon-specific dye-mediated transport we reacted some of the free amineson the surface of bovine serum albumin (BSA; Sigma) with Alexa488carboxylic acid succinimidyl ester (A488-CASE; Invitrogen). The couplingreaction was carried out for 4 hr at room temperature at neutral pH at amolar ratio of 10:1 A488-CASE:BSA at which point excess glycine wasadded to quench the reaction. The resulting purified product contained˜3 Alexa488 molecules per BSA molecule. Tetramethylrhodamineisothiocyanate (TRITC)-labeled 70 kDa dextan (Sigma) was used as aninternal control for filter damage. Fluorescence measurements were madeusing a BMG labtech FLUOstar Omega instrument set at 540 nm excitationand 610 nm emission for TRITC-Dextran (optimal Ex=547 and Em=572) and480 nm excitation and 520 nm emission for Alexa488 proteins (optimalEx=496 and Em=519).

Trans-epithelial transport flux rates were measured in vitro in theapical (Ap) to basolateral (BI) and the BI to Ap directions usingpolarized monolayers of Caco-2 cells to describe mucosal to serosal andserosal to mucosal flux events, respectively. Just prior to initiationof a transport study, the transepithelial resistance (TEER) of eachfilter was measured; monolayers TEER reading of <200 Ω·cm2 were excludedfrom the study. Ap and BI media was removed from included monolayers andthese surfaces were washed with once with phosphate buffered saline(PBS). One set of monolayers then received an Ap (donor) application of100 μL PBS containing 10 μg ntCholix-A488 and 10 μg TRITC-Dextran or 10μg BSA-A488 and 10 μg TRITC-Dextran. Receiver (BI) compartments thenreceived 500 μL PBS to set the T₀ for the transport study. Both donorand receiver compartments were sampled after 4 hr of incubation at 37°C. to determine the amount of material transported across the monolayerand the amount retained at the apical surface, respectively.

After 4 hour of exposure we observed ˜5% of the material added to theapical surface of these monolayers to be transported (see FIG. 2). Anyfilters showing levels of 75 kDa TRITC-Dextran in the basal compartmentwere excluded from the analysis. A control protein of BSA-Alexa488failed to show any significant levels in the basal compartment over thissame 4 hr period (see FIG. 2). The averages of transport were5.025±1.13% for Cholix and 0.56±0.33 for BSA (N=4). This dataestablishes that a genetically detoxified form of Cholix can efficientlytransport in vitro across polarized monolayers of a human intestinalcancer cell line, Caco-2.

EXAMPLE 3

Also prepared and expressed in E. coli. was a variant of Cholixtruncated at amino acid A³⁸⁶ (Cholix³⁸⁶) as well as a genetic ligationof green fluorescent protein (GFP) at the C-terminus of Cholix³⁸⁶(Cholix³⁸⁶GFP). Protein expression was achieved using E. coli DH5α cells(Invitrogen, Carlsbad, Calif.) following transformation by heat-shock (1min at 42° C.) with the appropriate plasmid. Transformed cells, selectedon antibiotic-containing media, were isolated and grown in Luria-Bertanibroth (Difco). Protein expression was induced by addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTGinduction, cells were harvested by centrifugation at 5,000×g for 10 minat 4° C. Inclusion bodies were isolated following cell lysis andproteins were solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0)plus 65 mM dithiothreitol. Following refolding and purification,proteins were stored at ˜5 ml/ml in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺at −80° C. Cholix³⁸⁶GFP refolding was monitored by acquisition andretention of the fluorescence signature associated with this fluorescentprotein; Sample et al., Chem Soc Rev, 38(10): p. 2852-64 (2009). Greenfluorescent protein (GFP) was purchased from Upstate (Charlottesville,Va.). All proteins used in these studies were confirmed to be at >90%purity based upon size exclusion chromatography.

Polystyrene beads (10 nm diameter) containing a covalently integratedred fluorescent dye with excitation/emission properties of 468/508 nmand having aldehyde surface functional groups (XPR-582) were obtainedfrom Duke Scientific (Palo Alto, Calif.). One hundred μl of XPR-582beads (at 2% solids) was mixed with approximately 2.5 nmoles GFP orCholix³⁸⁶GFP in a final volume of 200 μl neutral (pH 7.0) phosphatebuffered saline (PBS). After 2 hr of gentle rocking at room temperature,20 μl of a 2 mg/ml solution of bovine serum albumin (BSA; Sigma, St.Louis, Mo.) in PBS was added. Preparations were then dialyzed by threecycles of dilution with PBS and concentration using a 100,000 molecularweight cutoff Microcon filter device from Millipore (Bedford, Mass.).Final preparations of coated beads were at 1% solids.

EXAMPLE 4

A549 (ATCC CCL-185™), L929 (ATCC CRL-2148™), and Caco-2 (ATCC HTB-37™)cells were maintained in 5% CO₂ at 37° C. in complete media: Dulbecco'smodified Eagle's medium F12 (DMEM F12) supplemented with 10% fetalbovine serum, 2.5 mM glutamine, 100 U of penicillin/ml, and 100 μg ofstreptomycin/ml (Gibco BRL, Grand Island, N.Y.). Cells were fed every 2to 3 days with this media (designated complete medium) and passagedevery 5 to 7 days. For assays, cells were seeded into 24- or 96-wellplates and grown to confluence.

Caco-2 cells were grown as confluent monolayers on collagen-coated0.4-μm pore size polycarbonate membrane transwell supports(Corning-Costar, Cambridge, Mass.) and used 18-25 days after attaining atrans-epithelial electrical resistance (TEER) of >250 Ω·cm2 as measuredusing a chopstick Millicell-ERS® voltmeter (Millipore). Apical tobasolateral (A→B) and basolateral to apical (B→A) transport of Cholix orCholix³⁸⁶GFP across these monolayer was determined by measuring theamount of transported protein 4 hr after a 20 μg application at 37° C.TEER measurements and the extent of 10 kDa fluorescent dextran (measuredusing an HPLC size exclusion protocol) were used to verify monolayerbarrier properties during the course of the study. The extent of Cholixtransport was determined by titration of collected media in thecell-based cytotoxicity assay. Transported Cholix³⁸⁶GFP was measured byenzyme linked immunosorbant assay (ELISA) using anti-GFP antibody forcapture and the polyclonal sera to Cholix for detection.

Transport rates across polarized Caco-2 cells monolayers in vitro werecomparable for Cholix, ntCholix and Cholix³⁸⁶GFP as assess by ELISAformat analysis. In the case of Cholix, polarized Caco-2 cells were notintoxicated by the protein when examined for TUNEL detection ofapoptosis or lactate dehydrogenase (LDH) release. Importantly, Cholixand Cholix-based protein chimeras were found to transport efficientlyfrom the apical to basolateral surface of Caco-2 monolayers but not inthe basolateral to apical direction. These transport rates anddirectionality were comparable to that previously observed for PE testedin this same format. Additionally, we observed that addition rabbitanti-Cholix antisera failed to block the effective transport of Cholixor Cholix-related proteins across Caco-2 monolayers in vitro.

Confocal fluorescence microscopy was used to examine the nature ofCholix³⁸⁶GFP transcytosis across Caco-2 monolayers in vitro. A timecourse study showed Cholix³⁸⁶GFP entering into epithelial cells within 5minutes of its apical application and transporting through cells to thebasolateral region of the cell within 15 minutes. In samples exposed toapical Cholix³⁸⁶GFP for 15 minutes with subsequent removal of excessCholix³⁸⁶GFP from the apical chamber, GFP fluorescence was observed tocontinue in the direction of the basolateral surface of the cell and notback toward the apical surface. This unidirectional movement ofCholix³⁸⁶GFP was confirmed by measuring Cholix³⁸⁶GFP content in theapical and basolateral compartments over this time course. Applicationof Cholix³⁸⁶GFP at the basolateral surface of Caco-2 monolayers did notshow any significant fluorescence entering into the cells, consistentwith transport studies. Western blot analysis of transported Cholix,ntCholix and Cholix³⁸⁶GFP suggested that these proteins transportedwithout major alterations.

In vitro studies also showed that 100 nm diameter fluorescent latexbeads chemically coupled to Cholix³⁸⁶GFP efficiently transported acrossCaco-2 monolayers following an apical application. Latex bead selectionwith a 100 nm diameter provided a material that could readily fit withinthe lumen of a 125 nm diameter endosome derived from a clatherin-coatedpit. Thus, these studies suggest Cholix³⁸⁶GFP-latex beads to movethrough polarized Caco-2 cells by a mechanism consistent with endosomeuptake at the apical cell surface followed by endosome-basedintracellular trafficking. Pre-incubation of Cholix³⁸⁶GFP-coupled 100 nmdiameter fluorescent latex beads with anti-Cholix antisera failed toalter the transport of these beads. A similar amount of GFP chemicallycoupled to 100 nm diameter fluorescent latex beads did not facilitatethe in vitro transport of these particles across Caco-2 monolayers.Confocal fluorescence microscopy studies were consistent withdifferences observed for in transcytosis latex bead coated withCholix³⁸⁶GFP versus GFP.

The result that Cholix is capable of transporting across polarizedepithelial barriers similar to PE is unanticipated. While theirstructures are similar as suggested by crystallographic analysis, theirsurfaces amino acid composition is strikingly different; indeed,alignment methods based upon amino acid similarity would not readilymatch these two proteins. This is important in that the ability of apathogen-derived protein, such as these two virulence factors, tointeract with host cell receptors is presumed to involvesurface-expressed amino acid components. As both of these proteins (withtheir substantially different amino acid sequences) transportefficiently across polarized epithelia, it is highly likely that someother mechanism forms that basis for this transport capacity. It is ourcontention that the structural relationships shared by PE and Cholixforms the basis of the inherent capacity for their efficienttranscytosis. While both PE and Cholix proteins would have the capacityto bind to an apical surface receptor to gain access to endosomalcompartments it is more likely that this interaction and the potentialfor other receptors involved in the intracellular trafficking of theseproteins would be based upon conformational structures rather thanspecific amino acids on the protein surface.

EXAMPLE 5

In this Example, the preparation of a non-naturally occurring fusionmolecule as a single amino acid sequence and comprising a modifiedCholix toxin sequence, a cleavable linker sequence, and a biologicallyactive cargo, is generally described.

Seven exemplary fusion molecule expression vectors for delivering thepolypeptides interleukin-10 (SEQ ID NO: 82), interleukin-19 (SEQ ID NO:83), interleukin-20 (SEQ ID NO: 84), interleukin-22 (SEQ ID NO: 85),interleukin-24 (SEQ ID NO: 86), or interleukin-26 (SEQ ID NO: 87) areconstructed as generally described below. First, the polypeptide genesare amplified by PCR, incorporating restriction enzymes pairs of NdeIand EcoRI, PstI and PstI, AgeI and EcoRI, or PstI and EcoRI sites at twoends of the PCR products. After restriction enzyme digestion, the PCRproducts are cloned into an appropriate plasmid for cellular expression,which is digested with the corresponding restriction enzyme pairs. Theresulting constructs comprise a modified Cholix toxin comprising anamino acid sequence encoding amino acids 1-386 of SEQ ID NO: 1(Cholix³⁸⁶) and the respective polypeptides, and are also tagged with a6-His motif at the N-terminus of the polypeptide to facilitatepurification. The final plasmids are verified by restriction enzymedigestions and DNA sequencing.

Also prepared was a non-naturally occurring fusion molecule comprising aCholix⁴¹⁵ (SEQ ID NO: 52), a cleavable linker sequence having the aminoacid sequence set forth in SEQ ID NO: 121, and a biologically activecargo that is a IL-10 polypeptide consisting of amino acid residues20-178 of SEQ ID NO: 82 (this fusion molecule is designated“Cholix415-TEV-IL-10”, see FIG. 3 (SEQ ID NO: 122)), and a non-naturallyoccurring fusion molecule comprising a Cholix⁴¹⁵ (SEQ ID NO: 52), anon-cleavable linker sequence having the amino acid sequence set forthin SEQ ID NO: 98, and a biologically active cargo that is a IL-10polypeptide consisting of amino acid residues 20-178 of SEQ ID NO: 82(this fusion molecule is designated “Cholix⁴¹⁵-(G₄S)₃-IL-10”, see FIG. 3(SEQ ID NO: 123)).

Expression vectors comprising non-cleavable or cleavable linkers areconstructed by introducing sequences encoding the appropriate amino acidsequence. To do so, oligonucleotides that encode sequences complementaryto appropriate restriction sites and the amino acid sequence of thedesired linker are synthesized, then ligated into an expression vectorprepared as described above between the modified Cholix sequence and thepolypeptide sequence.

In various embodiments, the fusion molecules are expressed as follows:E. coli BL21(DE3) pLysS competent cells (Novagen, Madison, Wis.) aretransformed using a standard heat-shock method in the presence of theappropriate plasmid to generate fusion molecule expression cells,selected on ampicillin-containing media, and isolated and grown inLuria-Bertani broth (Difco; Becton Dickinson, Franklin Lakes, N.J.) withantibiotic, then induced for protein expression by the addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG) at OD 0.6. Two hours followingIPTG induction, cells are harvested by centrifugation at 5,000 rpm for10 min. Inclusion bodies are isolated following cell lysis and proteinsare solubilized in the buffer containing 100 mM Tris-HCl (pH 8.0), 2 mMEDTA, 6 M guanidine HCl, and 65 mM dithiothreitol. Solubilized fusionmolecule is refolded in the presence of 0.1 M Tris, pH=7.4, 500 mML-arginine, 0.9 mM GSSG, 2 mM EDTA. The refolded proteins are purifiedby Q sepharose Ion Exchange and Superdex 200 Gel Filtrationchromatography (Amersham Biosciences, Inc., Sweden). The purity ofproteins is assessed by SDS-PAGE and analytic HPLC (Agilent, Inc. PaloAlto, Calif.).

FIG. 4 is a ribbon diagram representation of an exemplary fusionmolecule, e.g., Cholix⁴¹⁵-TEV-IL-10 after refolding that would be drivenby IL-10 dimerization. IL-10 dimerization is envisaged to result inpurple Cholix⁴¹⁵/blue hIL-10 and orange Cholix⁴¹⁵/green organizationshown.

Cholix⁴¹⁵-TEV-IL-10 and Cholix⁴¹⁵-(G₄S)₃-IL-10 were evaluated to verifythe proper folding with regard to their anticipated molecular size.Following induction, expressed protein was collected from inclusionbodies. The extent of Cholix⁴¹⁵-TEV-IL-10 (depicted as “C” on the gel)expression and Cholix⁴¹⁵-(G₄S)₃-IL-10 (depicted as “N” on the gel)expression in inclusion bodies showed an apparent molecular weight of˜66 kDa that was comparable to the calculated mass of 66380.78 and65958.25 Daltons, respectively. See FIG. 5. The lack of these proteinsin supernatant media following inclusion body removal for the TEV linker(Cs) and non-TEV linker (Ns) are shown to demonstrate the extent andspecificity of chimera induction. SeeBlue® Plus2 Prestained MW standardsare shown.

EXAMPLE 6

This example describes in vitro methods to verify the proper folding ofthe fusion molecules with regard to their ability to carry abiologically active cargo across an intact epithelium.

The J774 mouse macrophage cell line can be used as an IL-10 responsivecell line (O'Farrell AM, et al., EMBO J, 17(4):1006-18, 1998). IL-10naturally forms a dimer that is required for its optimal activity.Cholix⁴¹⁵-(G₄S)₃-IL-10 expressed by E coli was collected from inclusionbodies and folded using a disulphide shuffle exchange buffer system. Theresulting material was purified by ion exchange and size exclusionchromatography that resulted in the isolation of a protein of ˜130 kDa,the anticipated size of an IL-10 dimer conjoined to two Cholix⁴¹⁵molecules (hereinafter “dimer Cholix⁴¹⁵-IL-10” fusion molecule). Thepreparation had a protein purity of ˜85-90% based upon SDS PAGE.Cultures of the J774.2 cell line were treated for 48 h with dimerCholix⁴¹⁵-IL-10 fusion molecule at concentrations of 25 nM and 250 nM.Compared to untreated matched cells, dimer Cholix⁴¹⁵-IL-10 fusionmolecule produced a dose-dependent decrease in cell number as assessedby flow cytometry of live/dead cells (see FIG. 6). Values representn=4±standard deviation.

Alternatively, one could co-culture the IL-10 responsive cells in thebasal compartment of the cell monolayers used for apical to basolateraltranscytosis (Rubas W, et al., Pharm Res. 13(1):23-6, 1996).

EXAMPLE 7

In this example, dimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule wasevaluated for its effect on the barrier properties of Caco-2 cellmonolayers in vitro. Caco-2 cells (a human colon cancer derived cellline) with media from the basolateral compartment being sampledperiodically for several hours (Rubas W, et al., J Pharm Sci.,85(2):165-9, 1996). Caco-2 (ATCC HTB-37™) cells are maintained in 5% CO₂at 37° C. in complete media: Dulbecco's modified Eagle's medium F12(DMEM F12) supplemented with 10% fetal bovine serum, 2.5 mM glutamine,100 U of penicillin/ml, and 100 μg of streptomycin/ml (Gibco BRL, GrandIsland, N.Y.). Cells are fed every 2 to 3 days with this media(designated complete medium) and passaged every 5 to 7 days. For assays,cells are seeded into 24- or 96-well plates and grown to confluence.

Established Caco-2 monolayers used for these studies had transepithelialelectrical resistance (TER) values of between ˜450-600 Ω·cm² (579 Ω·cm²average) as measured using a chopstick Millicell-ERS® voltmeter(Millipore). Fluorescein-labeled 70 kDa dextran and varyingconcentrations (4.7 nM, 23.6 nM and 236 nM) of dimerCholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule were added to the apical surfaceof these monolayers and the cumulative amount of florescence detected inthe basal compartment monitored over time by collecting 150 μL volumeswith replacement. As depicted in FIG. 7 and FIG. 8, in the absence ofCaco-2 cells on the filter support, the dextran rapidly moved from theapical to basal compartment. By comparison, the extent of 70 kDa dextrantransport was much less across Caco-2 monolayers and the various dimerCholix⁴¹⁵-IL-10 fusion molecules failed to have any dose-dependenteffect on the extent of 70 kDa dextran transport across these Caco-2monolayers and were not strikingly different from results obtained withCaco-2 monolayers not exposed to dimer Cholix⁴¹⁵-IL-10 fusion molecules.The dimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule does not overtly affectthe barrier properties of Caco-2 cell monolayers in vitro.

EXAMPLE 8

In this example, an ELISA assay is performed to evaluate the ability ofthe dimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule to move across Caco-2cell monolayers. A549 (ATCC CCL-185™), L929 (ATCC CRL-2148™), and Caco-2(ATCC HTB-37™) cells are maintained in 5% CO₂ at 37° C. in completemedia: Dulbecco's modified Eagle's medium F12 (DMEM F12) supplementedwith 10% fetal bovine serum, 2.5 mM glutamine, 100 U of penicillin/ml,and 100 μg of streptomycin/ml (Gibco BRL, Grand Island, N.Y.). Cells arefed every 2 to 3 days with this media (designated complete medium) andpassaged every 5 to 7 days. For assays, cells are seeded into 24- or96-well plates and grown to confluence.

Caco-2 cells are grown as confluent monolayers on collagen-coated 0.4-μmpore size polycarbonate membrane transwell supports (Corning-Costar,Cambridge, Mass.) and used 18-25 days after attaining a trans-epithelialelectrical resistance (TER) of >250 Ω·cm2 as measured using a chopstickMillicell-ERS® voltmeter (Millipore). Apical to basolateral (A→B)transport of dimer Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule across thesemonolayer is determined by measuring the amount of transported protein 4hr after a 4.7 nM, 23.6 nM and 236 nM application at 37° C. TERmeasurements and the extent of 10 kDa fluorescent dextran (measuredusing an HPLC size exclusion protocol) are used to verify monolayerbarrier properties during the course of the study. The extent of Cholixtransport is determined by titration of collected media in thecell-based cytotoxicity assay. Transported dimer Cholix⁴¹⁵-(G₄S)₃-IL-10fusion molecule is measured by enzyme linked immunosorbant assay (ELISA)using anti-IL-10 antibody for capture and the polyclonal sera to Cholixfor detection. As depicted in FIG. 9 (A and B), dimerCholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule moves across Caco-2 cellmonolayers.

EXAMPLE 9

In this Example, the preparation of a non-naturally occurring fusionmolecule that lacks a cleavable sequence is described. These fusionsmolecules are designed to specifically target the submucosal/GI spaceand limit the actions of the biologically active cargo to that space.

A plasmid construct is prepared encoding the non-toxic mutant form ofthe Cholix toxin, Cholix toxin ΔE581 (SEQ ID NO: 81). Protein expressionis achieved using E. coli DH5α cells (Invitrogen, Carlsbad, Calif.)following transformation by heat-shock (1 min at 42° C.) with theappropriate plasmid. Transformed cells, selected onantibiotic-containing media, are isolated and grown in Luria-Bertanibroth (Difco). Protein expression is induced by addition of 1 mMisopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTGinduction, cells are harvested by centrifugation at 5,000×g for 10 minat 4° C. Inclusion bodies are isolated following cell lysis and proteinsare solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0) plus 65 mMdithiothreitol. Following refolding and purification, proteins arestored at ˜5 ml/ml in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺ at −80° C. Allproteins used in these studies are confirmed to be at >90% purity basedupon size exclusion chromatography.

The Cholix toxin ΔE581 protein is then modified at its C-terminus toallow direct chemical coupling through a free sulfhydryl residue locatednear the C-terminus of the protein. The C-terminal modification includesa cysteine-constrained loop harboring the consensus cleavage sequencefor the highly selective protease from the tobacco etch virus (TEV), asecond cysteine, and a hexa-histadine (His₆) tag. The second Cys isincluded to form a disulphide bridge with the Cys ultimately used forcoupling. Adding the His₆ sequence to the protein simplifies thepurification and the TEV cleavage sequence provides a mechanism toselectively remove the terminal Cys residue following mild reduction.TEV cleavage and mild reduction with 0.1 mM dithiotheitol followingexpression and isolation of the ntCholix constructs allows for thedirect chemical coupling of a biologically active cargo via amaleimide-based reaction as a generic mechanism of cargo attachment.Following TEV protease cleavage, reduction, and cargo coupling through amaleimide reaction with the free sulfhydryl, removal of the freedC-terminal sequence was achieved by a second Ni²⁺ column chromatographystep.

EXAMPLE 10

Trans-epithelial transport of Cholix toxin ΔE581-cargo is assessed usingCaco-2 monolayers in vitro. Caco-2 cells (passage number 25-35) aregrown to confluent monolayers as previously described; Rubas, W. et al.,Pharm Res, 10:113-118 (1993). Briefly, cells are maintained at 37° C. inDMEM/high growth media enriched with 2 mM L-glutamine, 10% fetal bovineserum, and 100 Units of penicillin/streptomycin in an atmosphere of 5%CO₂ and 90% humidity. Cells are passaged every week at a split ratio of1:3 in 75cm² flasks and seeded onto prewetted and collagen-coatedpermeable (0.4 μm pore size) polycarbonate (Transwell™) filter supportsfrom Corning Costar (Cambridge, Mass.) at a density of 63,000 cells/cm².Growth media is replaced every other day. Confluent monolayers,determined by the acquisition of significant trans-epithelial resistance(TEER) determine using an volt-ohm-meter (World Precision Instruments,Sarasota, Fla.), are used 20-26 days post seeding.

Trans-epithelial transport flux rates are measured in vitro in theapical (Ap) to basolateral (BI) and the BI to Ap directions usingpolarized monolayers of Caco-2 cells to describe mucosal to serosal andserosal to mucosal flux events, respectively. Just prior to initiationof a transport study, the transepithelial resistance (TEER) of eachfilter is measured; monolayers TEER reading of <200 Ω·cm2 are excludedfrom the study. Ap and BI media is removed from included monolayers andthese surfaces are washed once with phosphate buffered saline (PBS). Oneset of monolayers then receives an Ap (donor) application of 100 μL PBScontaining 10 μg Cholix toxin ΔE581-cargo and 10 μg TRITC-Dextran or 10μg BSA-cargo and 10 μg TRITC-Dextran. Receiver (BI) compartments thenreceive 500 μL PBS to set the T₀ for the transport study. Both donor andreceiver compartments are sampled after 4 hr of incubation at 37° C. todetermine the amount of material transported across the monolayer andthe amount retained at the apical surface, respectively.

EXAMPLE 11

This example describes the preparation and expression in E. coli. of afusion molecule comprising a modified Cholix toxin comprising a sequenceencoding amino acids 1-415 of SEQ ID NO: 1 directly fused at itsC-terminus to an IL-10 polypeptide (referred to as a “Cholix⁴¹⁵-IL-10fusion molecule”). Protein expression is achieved using E. coli DH5αcells (Invitrogen, Carlsbad, Calif.) following transformation byheat-shock (1 min at 42° C.) with the appropriate plasmid. Transformedcells, selected on antibiotic-containing media, are isolated and grownin Luria-Bertani broth (Difco). Protein expression is induced byaddition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG). Two hoursfollowing IPTG induction, cells are harvested by centrifugation at5,000×g for 10 min at 4° C. Inclusion bodies are isolated following celllysis and proteins are solubilized in 6 M guanidine HCl and 2 mM EDTA(pH 8.0) plus 65 mM dithiothreitol. Following refolding andpurification, proteins are stored at ˜5 ml/ml in PBS (pH 7.4) lackingCa²⁺and Mg²⁺ at −80° C. All proteins used in these studies wereconfirmed to be at >90% purity based upon size exclusion chromatography.

Polystyrene beads (10 nm diameter) containing a covalently integratedred fluorescent dye with excitation/emission properties of 468/508 nmand having aldehyde surface functional groups (XPR-582) are obtainedfrom Duke Scientific (Palo Alto, Calif.). One hundred μl of XPR-582beads (at 2% solids) are mixed with approximately 2.5 nmoles IL-10 orCholix⁴¹⁵-IL-10 fusion molecule in a final volume of 200 μl neutral (pH7.0) phosphate buffered saline (PBS). After 2 hr of gentle rocking atroom temperature, 20 μl of a 2 mg/ml solution of bovine serum albumin(BSA; Sigma, St. Louis, Mo.) in PBS is added. Preparations are thendialyzed by three cycles of dilution with PBS and concentration using a100,000 molecular weight cutoff Microcon filter device from Millipore(Bedford, Mass.). Final preparations of coated beads were at 1% solids.

EXAMPLE 12

In this Example, non-naturally occurring isolated fusion moleculescomprising the modified Cholix toxin sequence of SEQ ID NO: 52(Cholix⁴¹⁵), a cleavable linker sequence (SEQ ID NO: 121) or anon-cleavable linker (SEQ ID NO: 98), and a biologically active cargothat is a TNFSF inhibitor, are prepared as described in Example 5, andevaluated as described in the Examples above to confirm proper folding,proper size,

Six exemplary fusion molecule expression vectors (3 for each linker)were prepared to test for the ability of the fusion molecules totransport apical to basal across epithelial cells a TNFSF inhibitorselected from: 1) a TNF inhibitor that is an antibody comprising theheavy chain variable region and light chain variable region sequences ofSEQ ID NO: 88 and 89; 2) a TNF inhibitor that is an antibody comprisingthe heavy chain variable region and light chain variable regionsequences of SEQ ID NO: 90 and 91; and 3) a TNFSF inhibitor that is adimer of a soluble human TNFR-p75 with the Fc portion of IgG comprisingthe sequence of SEQ ID NO: 92.

EXAMPLE 13

In this Example, non-naturally occurring isolated fusion moleculescomprising the modified Cholix toxin sequence of SEQ ID NO: 52(Cholix⁴¹⁵), a cleavable linker sequence (SEQ ID NO: 121) or anon-cleavable linker (SEQ ID NO: 98), and a biologically active cargothat is a glucose-lowering agent, are prepared as described in Example5, and evaluated as described in the Examples above to confirm properfolding, proper size,

Four exemplary fusion molecule expression vectors (2 for each linker)were prepared to test for the ability of the fusion molecules totransport apical to basal across epithelial cells a glucose-loweringagent selected from: 1) a GLP-1 agonist comprising the sequence of SEQID NO: 93; and 2) a GLP-1 agonist comprising the sequence of SEQ ID NO:94.

EXAMPLE 14

In this Example, non-naturally occurring isolated fusion moleculescomprising the modified Cholix toxin sequence of SEQ ID NO: 52(Cholix⁴¹⁵), a cleavable linker sequence (SEQ ID NO: 121) or anon-cleavable linker (SEQ ID NO: 98), and a biologically active cargothat is a human growth hormone, are prepared as described in Example 5,and evaluated as described in the Examples above to confirm properfolding, proper size,

Two exemplary fusion molecule expression vectors (one for each linker)were prepared to test for the ability of the fusion molecules totransport apical to basal across epithelial cells a human growth hormonecomprising the sequence of SEQ ID NO: 95.

EXAMPLE 15

This example describes histological detection in tissues of arepresentative biologically active cargo of the fusion moleculesprepared in Example 5. Following administration of a fusion molecule,animals are euthanized by CO₂ asphyxiation and exsanguinated by cardiacpuncture. Specific tissues (lymph nodes, trachea, brain, spleen liver,GI tract) are removed, briefly rinsed in PBS to remove any residualblood and frozen in OCT. Sections (5 microns thick) are placed ontoslides. Slides are fixed in acetone for 10 min and rinsed with PBS.Slides are incubated with 3% peroxidase for 5 min. Slides are thenblocked with protein for an additional 5 min. Primary antibody to therespective biologically active cargo is incubated onto slides for 30 minat a 1:100 dilution followed by PBS washes. Biotin-labeled secondaryantibody is then incubated for approximately 15 minutes followed by PBSwashes. Streptavidin HRP label is incubated onto slides for 15 minfollowed by PBS washes. HRP Chromagen is applied for 5 min followed byseveral rinses in distilled H20. Finally, the slides are counterstainedwith hematoxylin for 1 min, coverslipped, and examined for the presenceof the biologically active cargo.

The fusion molecules of the disclosure offer several advantages overconventional techniques for local or systemic delivery of macromoleculesto a subject. Foremost among such advantages is the ability to deliverthe biologically active cargo to a subject without using a needle topuncture the skin of the subject. Many subjects require repeated,regular doses of macromolecules. For example, diabetics must injectinsulin several times per day to control blood sugar concentrations.Such subjects' quality of life would be greatly improved if the deliveryof a macromolecule could be accomplished without injection, by avoidingpain or potential complications associated therewith.

In addition, coupling of the biologically active cargo to the remainderof the fusion molecule with a linker that is cleaved by an enzymepresent at a basolateral membrane of an epithelial cell allows thebiologically active cargo to be liberated from the fusion molecule andreleased from the remainder of the fusion molecule soon aftertranscytosis across the epithelial membrane. Such liberation reduces theprobability of induction of an immune response against the biologicallyactive cargo. It also allows the biologically active cargo to interactwith its target free from the remainder of the fusion molecule.

In addition, the non-naturally occurring fusion molecules which lack acleavable linker can be advantageous in that the anchoring effect of themodified Cholix toxin by its receptor(s) at the surface of, e.g., immunecells that also express the receptor for the biologically active cargo(but in considerably lower quantity) can allow for greater exposure ofthe biologically active cargo at the surface of the targeted cells, andprovide a synergistic effect via the binding of the Cholix to itsreceptor and, e.g., binding of IL-10 to the IL-10R.

Moreover, once transported across the GI epithelium, the fusionmolecules of the disclosure will exhibit extended half-life in serum,that is, the biologically active cargo of the fusion molecules willexhibit an extended serum half-life compared to the biologically activecargo in its non-fused state, and oral administration of the fusionmolecule can deliver a higher effective concentration of the deliveredbiologically active cargo to the liver of the subject than is observedin the subject's plasma.

Furthermore, the embodiments of the fusion molecules can be constructedand expressed in recombinant systems. Recombinant technology allows oneto make a fusion molecule having an insertion site designed forintroduction of any suitable biologically active cargo. Such insertionsites allow the skilled artisan to quickly and easily produce fusionmolecules for delivery of new biologically active cargo, should the needto do so arise.

All of the articles and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the articles and methods of this disclosure have beendescribed in terms of embodiments, it will be apparent to those of skillin the art that variations may be applied to the articles and methodswithout departing from the spirit and scope of the disclosure. All suchvariations and equivalents apparent to those skilled in the art, whethernow existing or later developed, are deemed to be within the spirit andscope of the disclosure as defined by the appended claims. All patents,patent applications, and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe disclosure pertains. All patents, patent applications, andpublications are herein incorporated by reference in their entirety forall purposes and to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference in its entirety for any and all purposes. The disclosureillustratively described herein suitably may be practiced in the absenceof any element(s) not specifically disclosed herein. Thus, for example,in each instance herein any of the terms “comprising”, “consistingessentially of”, and “consisting of” may be replaced with either of theother two terms. The terms and expressions which have been employed areused as terms of description and not of limitation, and there is nointention that in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure claimed. Thus, it should be understood thatalthough the present disclosure has been specifically disclosed byembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

SEQUENCE LISTINGS

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822.

SEQ ID NO: 1 is the 634 amino acid sequence of mature Vibrio choleraCholix toxin.

SEQ ID NO: 2 is a nucleic acid sequence encoding the 634 amino acidsequence mature V. cholera Cholix toxin.

SEQ ID NOs: 3-80 are the amino acid sequences of various truncatedCholix toxins derived from the mature Cholix toxin sequence set forth inSEQ ID NO: 1.

SEQ ID NO: 81 is the amino acid sequence of a mutated Cholix toxinwherein the amino acid residue E581 of SEQ ID NO: 1 has been deleted.

SEQ ID NO: 82 is the amino acid sequence of human interleukin-10(IL-10).

SEQ ID NO: 83 is the amino acid sequence of human interleukin-19(IL-19).

SEQ ID NO: 84 is the amino acid sequence of human interleukin-20(IL-20).

SEQ ID NO: 85 is the amino acid sequence of human interleukin-22(IL-22).

SEQ ID NO: 86 is the amino acid sequence of human interleukin-24(IL-24).

SEQ ID NO: 87 is the amino acid sequence of human interleukin-26(IL-26).

SEQ ID NO: 88—heavy chain variable region sequence for an anti-TNF-alphaantibody.

SEQ ID NO: 89—light chain variable region sequence for an anti-TNF-alphaantibody.

SEQ ID NO: 90—heavy chain variable region sequence for an anti-TNF-alphaantibody.

SEQ ID NO: 91—light chain variable region sequence for an anti-TNF-alphaantibody.

SEQ ID NO: 92—amino acid sequence of human TNFR-p75-Fc dimeric fusionprotein.

SEQ ID NO: 93—GLP-1 agonist peptide amino acid sequence (exenatide)

SEQ ID NO: 94—GLP-1 agonist peptide amino acid sequence (Liraglutide)

SEQ ID NO: 95—amino acid sequence of human growth hormone (somatotropin)

SEQ ID NOs: 96-121 are the amino acid sequences of various peptidelinkers

SEQ ID NO: 122 is the amino acid sequence of a Cholix⁴¹⁵-TEV-IL-10fusion molecule.

SEQ ID NO: 123 is the amino acid sequence of a Cholix⁴¹⁵-(G₄S)31L-10fusion molecule.

SEQUENCE LISTINGS SEQ ID NO: 1-mature Vibrio cholera Cholix toxin amino acid sequence VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELKSEQ ID NO: 2-nucleic acid sequence encoding the mature V. cholera Cholix toxin ATGGTCGAAGAAGCTTTAAACATCTTTGATGAATGCCGTTCGCCATGTTCGTTGACCCCGGAACCGGGTAAGCCGATTCAATCAAAACTGTCTATCCCTAGTGATGTTGTTCTGGATGAAGGTGTTCTGTATTACTCGATGACGATTAATGATGAGCAGAATGATATTAAGGATGAGGACAAAGGCGAGTCCATTATCACTATTGGTGAATTTGCCACAGTACGCGCGACTAGACATTATGTTAATCAAGATGCGCCTTTTGGTGTCATCCATTTAGATATTACGACAGAAAATGGTACAAAAACGTACTCTTATAACCGCAAAGAGGGTGAATTTGCAATCAATTGGTTAGTGCCTATTGGTGAAGATTCTCCTGCAAGCATCAAAATCTCCGTTGATGAGCTCGATCAGCAACGCAATATCATCGAGGTGCCTAAACTGTATAGTATTGATCTCGATAACCAAACGTTAGAGCAGTGGAAAACCCAAGGTAATGTTTCTTTTTCGGTAACGCGTCCTGAACATAATATCGCTATCTCTTGGCCAAGCGTGAGTTACAAAGCAGCGCAGAAAGAGGGTTCACGCCATAAGCGTTGGGCTCATTGGCATACAGGCTTAGCACTGTGTTGGCTTGTGCCAATGGATGCTATCTATAACTATATCACCCAGCAAAATTGTACTTTAGGGGATAATTGGTTTGGTGGCTCTTATGAGACTGTTGCAGGCACTCCGAAGGTGATTACGGTTAAGCAAGGGATTGAACAAAAGCCAGTTGAGCAGCGCATCCATTTCTCCAAGGGGAATGCGATGAGCGCACTTGCTGCTCATCGCGTCTGTGGTGTGCCATTAGAAACTTTGGCGCGCAGTCGCAAACCTCGTGATCTGACGGATGATTTATCATGTGCCTATCAAGCGCAGAATATCGTGAGTTTATTTGTCGCGACGCGTATCCTGTTCTCTCATCTGGATAGCGTATTTACTCTGAATCTTGACGAACAAGAACCAGAGGTGGCTGAACGTCTAAGTGATCTTCGCCGTATCAATGAAAATAACCCGGGCATGGTTACACAGGTTTTAACCGTTGCTCGTCAGATCTATAACGATTATGTCACTCACCATCCGGGCTTAACTCCTGAGCAAACCAGTGCGGGTGCACAAGCTGCCGATATCCTCTCTTTATTTTGCCCAGATGCTGATAAGTCTTGTGTGGCTTCAAACAACGATCAAGCCAATATCAACATCGAGTCTCGTTCTGGCCGTTCATATTTGCCTGAAAACCGTGCGGTAATCACCCCTCAAGGCGTCACAAATTGGACTTACCAGGAACTCGAAGCAACACATCAAGCTCTGACTCGTGAGGGTTATGTGTTCGTGGGTTACCATGGTACGAATCATGTCGCTGCGCAAACCATCGTGAATCGCATTGCCCCTGTTCCGCGCGGCAACAACACTGAAAACGAGGAAAAGTGGGGCGGGTTATATGTTGCAACTCACGCTGAAGTTGCCCATGGTTATGCTCGCATCAAAGAAGGGACAGGGGAGTATGGCCTTCCGACCCGTGCTGAGCGCGACGCTCGTGGGGTAATGCTGCGCGTGTATATCCCTCGTGCTTCATTAGAACGTTTTTATCGCACGAATACACCTTTGGAAAATGCTGAGGAGCATATCACGCAAGTGATTGGTCATTCTTTGCCATTACGCAATGAAGCATTTACTGGTCCAGAAAGTGCGGGCGGGGAAGACGAAACTGTCATTGGCTGGGATATGGCGATTCATGCAGTTGCGATCCCTTCGACTATCCCAGGGAACGCTTACGAAGAATTGGCGATTGATGAGGAGGCTGTTGCAAAAGAGCAATCGATTAGCACAAAACCACCTTATAAAGAGCGCAAAGATGAACTTAAGSEQ ID NO: 3-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ ASEQ ID NO: 4-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQSEQ ID NO: 5-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGASEQ ID NO: 6-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGSEQ ID NO: 7-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSASEQ ID NO: 8-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSSEQ ID NO: 9-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁰ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSEQ ID NO: 10-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQSEQ ID NO: 11-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPESEQ ID NO: 12-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPSEQ ID NO: 13-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTSEQ ID NO: 14-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLSEQ ID NO: 15- modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGSEQ ID NO: 16- modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPSEQ ID NO: 17- modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHSEQ ID NO: 18- modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHSEQ ID NO: 19-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁷⁰VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTSEQ ID NO: 20-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVSEQ ID NO: 21-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYSEQ ID NO: 22-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDSEQ ID NO: 23-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNSEQ ID NO: 24-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYSEQ ID NO: 25-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQISEQ ID NO: 26-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQSEQ ID NO: 27-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARSEQ ID NO: 28-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVASEQ ID NO: 29-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁶⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVSEQ ID NO: 30-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTSEQ ID NO: 31-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLSEQ ID NO: 32-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVSEQ ID NO: 33-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQSEQ ID NO: 34-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTSEQ ID NO: 35-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVSEQ ID NO: 36-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMSEQ ID NO: 37-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGSEQ ID NO: 38-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPSEQ ID NO: 39-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁵⁰VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNSEQ ID NO: 40-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁴⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENSEQ ID NO: 41-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁴⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINESEQ ID NO: 42-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENSEQ ID NO: 43-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPESEQ ID NO: 44-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPSEQ ID NO: 45-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLSEQ ID NO: 46-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²¹VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYSEQ ID NO: 47-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴²⁰ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSSEQ ID NO: 48-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSEQ ID NO: 49-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGSEQ ID NO: 50-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSSEQ ID NO: 51-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSEQ ID NO: 52-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESSEQ ID NO: 53-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESEQ ID NO: 54-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINISEQ ID NO: 55-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINSEQ ID NO: 56-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹¹VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANISEQ ID NO: 57-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴¹⁰VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANSEQ ID NO: 58-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQASEQ ID NO: 59-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQSEQ ID NO: 60-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDSEQ ID NO: 61-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNSEQ ID NO: 62-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSEQ ID NO: 63-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASSEQ ID NO: 64-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASEQ ID NO: 65-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVSEQ ID NO: 66-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCSEQ ID NO: 67-modified Vibrio cholera Cholix toxin amino acid sequence Cholix⁴⁰⁰ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSSEQ ID NO: 68-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSEQ ID NO: 69-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADSEQ ID NO: 70-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDA SEQ ID NO: 71-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁶ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDSEQ ID NO: 72-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁵ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPSEQ ID NO: 73-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁴ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCSEQ ID NO: 74-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹³ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AADILSLFSEQ ID NO: 75-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹² VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AADILSLSEQ ID NO: 76-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹¹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AADILSSEQ ID NO: 77-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁹⁰ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AADILSEQ ID NO: 78-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁹ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AADISEQ ID NO: 79-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁸ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AAD SEQ ID NO: 80-modified Vibrio cholera Cholix toxin amino acid sequence Cholix³⁸⁷ VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQ AASEQ ID NO: 81-modified Vibrio cholera Cholix toxin amino acid sequence CholixΔ581 VEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLDITTENGTKTYSYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSYETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDTVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELKSEQ ID NO: 82-human interleukin-10 amino acid sequence MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSEQ ID NO: 83-human interleukin-19 amino acid sequence MKLQCVSLWLLGTILILCSVDNHGLRRCLISTDMHHIEESFQEIKRAIQAKDTFPNVTILSTLETLQIIKPLDVCCVTKNLLAFYVDRVFKDHQEPNPKILRKISSIANSFLYMQKTLRQCQEQRQCHCRQEATNATRVIHDNYDQLEVHAAAIKSLGELDVFLAWINKNHEVMSSASEQ ID NO: 84-human interleukin-20 amino acid sequenceMKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSCVIATNLQEIRNGFSEIRGSVQAKDGNIDIRILRRTESLQDTKPANRCCLLRHLLRLYLDRVFKNYQTPDHYTLRKISSLANSFLTIKKDLRLCHAHMTCHCGEEAMKKYSQILSHFEKLEPQAAVVKALGELDILLQWMEETESEQ ID NO: 85-human interleukin-22 amino acid sequence MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACISEQ ID NO: 86-human interleukin-24 amino acid sequenceMNFQQRLQSLWILASRPFCPPLLATASQMQMVVLPCLGFILLLWSQVSGAQGQEFHFGPCQVKGVVPQKLWEAFWAVKDTMQAQDNITSARLLQQEVLQNVSDAESCYLVHTLLEFYLKTVFKNYHNRTVEVRTLKSFSTLANNFVLIVSQLQPSQENEMFSIRDSAHRRFLLFRRAFKQLDVEAALTKALGEVDILLTWMQKFYKLSEQ ID NO: 87-human interleukin-26 amino acid sequence MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKLLESSQSEQ ID NO: 88-heavy chain variable region sequence for an anti-TNF-alpha antibody EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVERGFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSEQ ID NO: 89-light chain variable region sequence for an anti-TNF-alpha antibody DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 90-heavy chain variable region sequence for an anti-TNF-alpha antibody QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAIISFDGSNKSSADSVKGRFTYSRRNSKNALFLQMNSLRAEDTAVFYCARDRGVSAGGNYYYYGMDVWGQGTTVT VSS SEQ ID NO: 91-light chain variable region sequence for an anti-TNF-alpha antibody EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTRFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDILSEQ ID NO: 92-amino acid sequence of human TNFR-p75-Fc dimeric fusion protein LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 93-GLP-1 agonist peptide amino acid sequence (exenatide) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSSEQ ID NO: 94-GLP-1 agonist peptide amino acid sequence (Liraglutide) HAEGTFTSDVSSYLEGQAAKEEFIIAWLVKGRGSEQ ID NO: 95-amino acid sequence of human growth hormone (somatotropin) FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNRE ETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLED GSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF SEQ ID NO: 96-amino acid sequence of a peptide linker  GGGGS SEQ ID NO: 97-amino acid sequence of a peptide linker  GGGGSGGGGS SEQ ID NO: 98-amino acid sequence of a peptide linker  GGGGSGGGGSGGGGS SEQ ID NO: 99-amino acid sequence of a peptide linker  GGGGSGGG SEQ ID NO: 100-amino acid sequence of a peptide linker  AAPF SEQ ID NO: 101-amino acid sequence of a peptide linker  GGF SEQ ID NO: 102-amino acid sequence of a peptide linker  AAPV SEQ ID NO: 103-amino acid sequence of a peptide linker  GGL SEQ ID NO: 104-amino acid sequence of a peptide linker  AAL SEQ ID NO: 105-amino acid sequence of a peptide linker  FVR SEQ ID NO: 106-amino acid sequence of a peptide linker  VGR SEQ ID NO: 107-amino acid sequence of a peptide linker  RKPR SEQ ID NO: 108-amino acid sequence of a peptide linker  Y VAD Xaa Xaa =any amino acid  SEQ ID NO: 109-amino acid sequence of a peptide linker D Xaa Xaa D Xaa Xaa = any amino acid SEQ ID NO: 110-amino acid sequence of a peptide linker R (Xaa)_(n) R Xaa Xaa = any amino acid n = 0, 2, 4 or 6 SEQ ID NO: 111-amino acid sequence of a peptide linker K (Xaa)_(n) R Xaa Xaa = any amino acid n = 0, 2, 4 or 6 SEQ ID NO: 112-amino acid sequence of a peptide linker E R T K R Xaa Xaa = any amino acid SEQ ID NO: 113-amino acid sequence of a peptide linker R V R R Xaa Xaa = any amino acid SEQ ID NO: 114-amino acid sequence of a peptide linker Decanoyl-R V R R Xaa Xaa = any amino acid SEQ ID NO: 115-amino acid sequence of a peptide linker P Xaa W V P Xaa Xaa = any amino acid SEQ ID NO: 116-amino acid sequence of a peptide linker  W V A Xaa Xaa =any amino acid  SEQ ID NO: 117-amino acid sequence of a peptide linker Xaa F Xaa Xaa Xaa = any amino acid SEQ ID NO: 118-amino acid sequence of a peptide linker Xaa Y Xaa Xaa Xaa = any amino acid n = 0,2, 4 or 6 SEQ ID NO: 119-amino acid sequence of a peptide linker Xaa W Xaa Xaa Xaa = any amino acid n = 0, 2, 4 or 6 SEQ ID NO: 120-amino acid sequence of a peptide linker DRW IP FHLL in combination with (V, A or P)-Y-(S, P or A) SEQ ID NO: 121-amino acid sequence of a peptide linker  GGGGSGGGENLYFQS SEQ ID NO: 122-amino acid sequence of a Cholix⁴¹⁵-TEV-IL-10 fusion molecule MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESGGGGSGGGENLYFQSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNSEQ ID NO: 123-amino acid sequence of a Cholix⁴¹⁵-(G₄S)₃-IL-10 fusion molecule MVEEALNIFDECRSPCSLTPEPGKPIQSKLSIPGDVVLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVSQDAPFGVINLDITTENGTKTYSFNRKESEFAINWLVPIGEDSPASIKISIDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSVTRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPIDAIYNYITQQNCTLGDNWFGGSYETVAGTPKAITVKQGIEQKPVEQRIHFSKKNAMEALAAHRVCGVPLETLARSRKPRDLPDDLSCAYNAQQIVSLFLATRILFTHIDSIFTLNLDGQEPEVAERLDDLRRINENNPGMVIQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNSDQANINIESGGGGSGGGGSGGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

What is claimed is:
 1. A pharmaceutical composition comprising anon-naturally occurring fusion molecule and one or more pharmaceuticallyacceptable carriers, formulated for oral delivery to a subject, whereinthe fusion molecule comprises a modified Cholix toxin coupled to abiologically active cargo, wherein the modified Cholix toxin is mutatedat an amino acid residue within Cholix toxin domain III that renders theCholix toxin non-toxic, and wherein the fusion molecule has the abilityto activate the receptor for the biologically active cargo, or to enablethe catalytic process of a catalytically-active material.
 2. Thepharmaceutical composition of claim 1, wherein the modified Cholix toxincomprises the amino acid sequence set forth in SEQ ID NO:
 81. 3. Thepharmaceutical composition according to claim 1, wherein thebiologically active cargo is a macromolecule, small molecule, peptide,polypeptide, nucleic acid, mRNA, miRNA, shRNA, siRNA, antisensemolecule, antibody, DNA, plasmid, vaccine, polymer nanoparticle, orcatalytically-active material.
 4. The pharmaceutical compositionaccording to claim 1, wherein the modified Cholix toxin is directlycoupled to the biologically active cargo.
 5. The pharmaceuticalcomposition of claim 4, wherein the biologically active cargo isdirectly coupled to the C-terminus of the modified Cholix toxin.
 6. Thepharmaceutical composition according to claim 1, wherein the modifiedCholix toxin is chemically coupled to the biologically active cargo. 7.The pharmaceutical composition according to claim 1, wherein themodified Cholix toxin is coupled to the biologically active cargo by anon-cleavable linker, wherein the modified Cholix targets said cargo tospecific cells, including cells of the immune system such asmacrophages, antigen-presenting cells and dendritic cells.
 8. Thepharmaceutical composition according to claim 1, wherein the modifiedCholix toxin is coupled to the biologically active cargo by a cleavablelinker.
 9. A method for delivering a therapeutic cargo to a subject, themethod comprising orally administering to the subject a pharmaceuticalcomposition comprising a non-toxic Cholix toxin coupled to a therapeuticcargo.
 10. The method of claim 9, wherein the modified Cholix toxincomprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ IDNO:
 81. 11. The method of claim 9, wherein the biologically active cargois a macromolecule, small molecule, peptide, polypeptide, nucleic acid,mRNA, miRNA, shRNA, siRNA, antisense molecule, antibody, DNA, plasmid,vaccine, polymer nanoparticle, or catalytically-active material.
 12. Amethod of claim 9, wherein the modified Cholix toxin is directly coupledto the biologically active cargo.
 13. The method of claim 9, wherein thebiologically active cargo is directly coupled to the C-terminus of themodified Cholix toxin.
 14. The method of claim 9, wherein the modifiedCholix toxin is chemically coupled to the biologically active cargo. 15.The method of claim 9, wherein the modified Cholix toxin is coupled tothe biologically active cargo by a non-cleavable linker, wherein themodified Cholix targets said cargo to specific cells, including cells ofthe immune system such as macrophages, antigen-presenting cells anddendritic cells.
 16. The method of claim 9, wherein the modified Cholixtoxin is coupled to the biologically active cargo by a cleavable linker.